Pichinde virus reverse genetics systems and methods of use

ABSTRACT

Provided herein are genetically engineered Pichinde viruses that include three ambisense genomic segments. The first genomic segment includes a coding region encoding a Z protein and a coding region encoding a L RdRp protein. The second genomic segment includes a coding region encoding a nucleoprotein (NP) and the third genomic segment includes a coding region encoding a glycoprotein. Each of the second and third genomic segments optionally include an additional coding region that may encode an antigen or a detectable marker. Also provided herein is a reverse genetics system for making a genetically engineered Pichinde virus, and a collection of vectors that can be used to produce a genetically engineered Pichinde virus. Further provided are methods for using a reverse genetics system, and methods for producing an immune response in a subject.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/513,045, filed Mar. 21, 2017 (now issued as U.S. Pat. No.10,533,159), which is the § 371 U.S. National Stage of InternationalApplication No. PCT/US2015/051337, filed Sep. 22, 2015, which claims thebenefit of U.S. Provisional Application No. 62/053,443, filed Sep. 22,2014, the disclosures of which are incorporated by reference herein.

GOVERNMENT FUNDING

This invention was made with government support under AI083409 awardedby the National Institutes of Health. The government has certain rightsin the invention.

SEQUENCE LISTING

This application contains a Sequence Listing electronically submitted tothe United States Patent and Trademark Office via EFS-Web as an ASCIItext file entitled “2015-09-22-SequenceListing_ST25.txt” having a sizeof 45 KB and created on Sep. 22, 2015. Due to the electronic filing ofthe Sequence Listing, the electronically submitted Sequence Listingserves as both the paper copy required by 37 CFR § 1.821(c) and the CRFrequired by § 1.821(e). The information contained in the SequenceListing is incorporated by reference herein

BACKGROUND

Arenavirus family includes a group of bi-segmented enveloped RNA viruseswith genomes encoding a total of four genes in opposite orientation(Buchmeier et al., 2007, p. 1791-1827. In Knipe D M, Howley P M (ed.),Fields Virology, 5th ed. Lippincott Williams & Wilkins, Philadelphia,Pa.). The Z protein produced from the large (L) genomic segment is asmall RING-domain containing matrix protein that mediates virus buddingand also regulates viral RNA synthesis. The large L protein (˜200 kDa)encoded also on the L segment is the RNA-dependent RNA polymerase (RdRp)protein that is required for viral RNA synthesis. The glycoprotein (GPC)encoded on the small (S) segment is post-translationally processed intoa stable signal peptide (SSP), the receptor-binding G1 protein, and thetransmembrane G2 protein. The nucleoprotein (NP) of the S segmentencapsidates viral genomic RNAs, is required for viral RNA synthesis,and also suppresses host innate immune responses.

Arenaviruses are zoonotic RNA viruses with rodents as their primarynatural hosts (for a review, see McLay et al., 2014, J Gen Virol 95(Pt1):1-15). Humans can be infected with arenaviruses through directcontact of skin lesions with rodent excretions, eating foodscontaminated with rodent excretions, or inhalation of tainted aerosols.Only a few arenaviruses can cause diseases in humans (for a review, seeMcLay et al., 2013, Antiviral Res 97:81-92). For example, lymphocyticchoriomeningitis virus (LCMV) can cause central nerve system diseases,whereas Lassa virus (LASV) and several other arenaviruses can causehemorrhagic fevers that can potentially result in terminal shock anddeath. Limited therapeutic options are available for treating theseviral infections. The only available antiviral drug (ribavirin) showssome beneficial effects but it has many side effects and must beadministered soon after the infection when the disease is oftenmis-diagnosed as the symptoms are only flu-like and insidious.

Due to the high containment requirement (BSL-4) to work with pathogenicarenaviruses and the cost associated with working with non-humanprimates, only limited vaccine studies have been done. There arecurrently no vaccines for these pathogenic arenaviruses, except for theCandid #1 that is not FDA-approved but is available only for off-labeledusage against Junin virus, which causes Argentine hemorrhagic fever.Several safe and convenient systems have been developed to studyarenavirus replication and pathogenesis in the conventional BSL-2laboratory, such as the Pichinde virus model system (Lan et al., 2009, JVirol 83:6357-6362, Liang et al., 2009, Ann NY Acad Sci 1171 Suppl1:E65-74, Kumar et al., 2012, Virology 433:97-103, Wang et al., 2012, JVirol 86:9794-9801, McLay et al., 2013, J Virol 87:6635-6643). Pichindevirus (PICV) was isolated from rice rats in Columbia. It does not causedisease in humans, but can cause hemorrhagic fever-like symptoms inguinea pigs and thus is an ideal surrogate model to studyarenavirus-induced hemorrhagic fevers. Serological evidence suggests avery low seroprevalence in humans (2 out of 82 people living or workingin close association with habitats of infected rodents and 6 out of 13laboratory workers have shown anti-PICV serum positivity but no distinctillnesses (Trapido et al., 1971, Am J Trop Med Hyg 20:631-641, Buchmeieret al., 1974, Infect Immun 9:821-823). Therefore, there is little to nopreexisting immunity against PICV in the general human population, incontrast to LCMV, a prototypic arenavirus, which shows 2-5%seroprevalence in humans.

SUMMARY OF THE APPLICATION

Provided herein is a genetically engineered Pichinde virus. In oneembodiment, the virus includes three ambisense genomic segments. Thefirst genomic segment includes a coding region encoding a Z protein anda coding region encoding a L RdRp protein. The second genomic segmentincludes a coding region encoding a nucleoprotein (NP) and a firstrestriction enzyme site. The third genomic segment includes a codingregion encoding a glycoprotein and a second restriction enzyme site. Inone embodiment, the NP protein includes an amino acid sequence having atleast 80% identity to SEQ ID NO:3. In one embodiment, nucleoproteinincludes at least one mutation that reduces the exoribonuclease activityof the nucleoprotein, wherein the mutation is selected from an asparticacid at about amino acid 380, a glutamic acid at about amino acid 382,an aspartic acid at about amino acid 525, a histidine at about aminoacid 520, and an aspartic acid at about amino acid 457, wherein theaspartic acid, the glutamic acid, or the histidine is substituted withany other amino acid. In one embodiment, the glycoprotein includes atleast one mutation that alters the activity of the glycoprotein, whereinthe mutation is selected from an asparagine at about amino acid 20 andan asparagine at about amino acid 404, and wherein the asparagine issubstituted with any other amino acid. D, E, or H is substituted withany other amino acid.

In one embodiment, the second genomic segment includes a multiplecloning site, and the first restriction enzyme site is part of themultiple cloning site. The second genomic segment may further include acoding region encoding a first protein inserted at the first restrictionsite. In one embodiment, the third genomic segment includes a multiplecloning site, and the second restriction enzyme site is part of themultiple cloning site. In one embodiment, the third genomic segment mayfurther include a coding region encoding a second protein inserted atthe first restriction site. In one embodiment, the second genomicsegment further includes a coding region encoding a first proteininserted at the first restriction site, and the third genomic segmentfurther includes a coding region encoding a second protein inserted atthe first restriction site. In one embodiment, the first protein and thesecond protein are selected from an antigen and a detectable marker. Inone embodiment, the antigen is a protein expressed by a viral pathogen,a prokaryotic pathogen, or a eukaryotic pathogen. The first protein andthe second protein may be the same or may be different.

Also provided herein is a collection of vectors. In one embodiment, thevectors are plasmids. In one embodiment, the collection includes a firstvector encoding a first genomic segment including a coding regionencoding a Z protein and a coding region encoding a L RdRp protein,wherein the first genomic segment is antigenomic, a second vectorencoding a second genomic segment including a coding region encoding anucleoprotein (NP) and a first restriction enzyme site, wherein thesecond genomic segment is antigenomic, and a third vector encoding athird genomic segment includes a coding region encoding a glycoproteinand a second restriction enzyme site, wherein the third genomic segmentis antigenomic. In one embodiment, the NP protein includes an amino acidsequence having at least 80% identity to SEQ ID NO:3.

In one embodiment, the second genomic segment includes a multiplecloning site, and the first restriction enzyme site is part of themultiple cloning site. The second genomic segment may further include acoding region encoding a first protein inserted at the first restrictionsite. In one embodiment, the third genomic segment includes a multiplecloning site, and the second restriction enzyme site is part of themultiple cloning site. In one embodiment, the third genomic segment mayfurther include a coding region encoding a second protein inserted atthe first restriction site. In one embodiment, the second genomicsegment further includes a coding region encoding a first proteininserted at the first restriction site, and the third genomic segmentfurther includes a coding region encoding a second protein inserted atthe first restriction site. In one embodiment, the first protein and thesecond protein are selected from an antigen and a detectable marker. Inone embodiment, the antigen is a protein expressed by a viral pathogen,a prokaryotic pathogen, or a eukaryotic pathogen. The first protein andthe second protein may be the same or may be different.

Further provided are methods. In one embodiment, a method includesmaking a genetically engineered Pichinde virus as described herein. Themethod includes introducing into a cell the collection of vectorsdescribed herein, and incubating the cells in a medium under conditionssuitable for expression and packaging of the first, second, and thirdgenomic segments into a virus particle. The method may also includeisolating an infectious virus particle from the medium.

Also provided herein is a reverse genetics system for making agenetically engineered Pichinde virus, wherein the system includes threevectors. The first vector encodes a first genomic segment including acoding region encoding a Z protein and a coding region encoding a L RdRpprotein, wherein the first genomic segment is antigenomic, the secondvector encodes a second genomic segment including a coding regionencoding a nucleoprotein (NP) and a first restriction enzyme site,wherein the second genomic segment is antigenomic, and the third vectorencodes a third genomic segment includes a coding region encoding aglycoprotein and a second restriction enzyme site, wherein the thirdgenomic segment is antigenomic. In one embodiment, the NP proteinincludes an amino acid sequence having at least 80% identity to SEQ IDNO:3.

In one embodiment, a method includes using a reverse genetics system,including introducing into a cell three vectors of genomic segmentsdescribed herein, incubating the cell under conditions suitable for thetranscription of the three genomic segments and expression of the codingregions of each genomic segment. In one embodiment, the method alsoincludes isolating infectious virus particles produced by the cell,wherein each infectious virus particle includes the three genomicsegments. In one embodiment, the introducing includes transfecting acell with the three genomic segments. In one embodiment, the introducingincludes contacting the cell with an infectious virus particle includingthe three genomic segments. In one embodiment, the cell is ex vivo, suchas a vertebrate cell. In one embodiment, the vertebrate cell may be amammalian cell, such as a human cell, or the vertebrate cell may be anavian cell, such as a chicken embryonic fibroblast.

In one embodiment, a method includes producing an immune response in asubject. The method includes administering to a subject an infectiousvirus particle described herein, wherein the second genomic segmentincludes a coding region encoding a first antigen, and the third genomicsegment further includes a coding region encoding a second antigen. Inone embodiment, the cell is ex vivo, such as a vertebrate cell. In oneembodiment, the vertebrate cell may be a mammalian cell, such as a humancell, or the vertebrate cell may be an avian cell. The immune responsemay include a humoral immune response, a cell-mediated immune response,or a combination thereof. In one embodiment, the antigen is a proteinexpressed by a viral pathogen, a prokaryotic pathogen, or a eukaryoticpathogen, or a fragment thereof. The subject may have been exposed to,or is at risk of exposure to the viral pathogen, the prokaryoticpathogen, or the eukaryotic pathogen. In one embodiment, theadministering includes administering at least two populations ofinfectious virus particles, wherein each population of infectious virusparticle encodes a different antigen.

Also provided herein is an infectious virus particle as describedherein, and a composition that includes an infectious virus particledescribed herein.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Diagram of the 2-plasmid PICV reverse genetics system (Lan etal., 2009, J Virol 83:6357-6362).

FIG. 2. The 3-plasmid system to generate infectious tri-segmentedPICV-rP18tri-GFP.

FIG. 3. Characterization of rP18tri-GFP virus. A549 or BHK-21 cells wereinfected with rP18tri-GFP virus for 24 hours, and GFP expression wasdetected in the infected cells. Virus supernatants were harvested andused to infect a fresh culture of the BHK-21 cells, in which GFPexpression was also detected.

FIG. 4. Virus growth comparison of rP2, rP18, tri-segmented virusesrP18tri-GFP and rP18tri-RLuc in BHK-21 cell at high (moi=0.5) and low(moi=0.01) moi.

FIG. 5. Generation of tri-segmented PICV carrying influenza HA or NPantigen.

FIG. 6. Detection of expression of the GFP reporter gene product and theinfluenza HA or NP proteins that were expressed from the rP18tri-basedvaccine vectors.

FIG. 7. The rP18tri-based influenza vaccines confer protective immunityin mice. 1e5, 1×10⁵; 2e4, 2×10⁴; IP, intraperitoneal.

FIG. 8. HAI titers induced by the rP18tri-based influenza vaccines.

FIG. 9. HAI titers induced by the rP18tri-GFP/HA virus via differentroutes of vaccination. IN, intranasal; IM, intramuscular; IP,intraperitoneal.

FIG. 10. Protection conferred by rP18tri-GFP/HA following differentroutes of vaccination.

FIG. 11. Analysis of the NP-specific CTL responses by NP tetrameranalysis.

FIG. 12. Kinetics of CD8+ T cell responses elicited following differentroutes of vaccination.

FIG. 13. Vaccine conferred long lasting immunity and protection. C57BL6mice were challenged with 10LD₅₀ A/PR/8 after 1 or 2 months of boostingwith the rP18tri-GFP/HA vaccine vector. A. HI titres at day 14 and day30 post prime (dpp), day 30 and day 60 post boost (dpb). B. Viral titrein lungs at 6 dpi. C. Percent weight loss. D. H&E stained lung sections.

FIG. 14. Protection conferred by priming with the rP18tri-GFP/HA vaccinevector. A. Neutralizing antibody titres following different dosages ofthe rP18tri-GFP/HA vaccination. B. Percent weight loss. C. H&E stainedlung sections.

FIG. 15. Plaque size and expression of HA and NP from the rP18tri-HA/NPand the rP18tri-NP/HA

FIG. 16. T cell and humoral responses induced by vector expressing dualantigens (HA and NP). A. Representative FACS plots for NP₃₃₆ tetramer⁺CD44⁺ CD8⁺T cells in mouse blood at day 7 post-vaccination. B.Frequencies of NP specific CD8⁺ T cells in the peripheral blood tested 7days post priming and 7 days after boosting by tetramer staining. C.Viral neutralizing antibody titres were determined using HI assay ofsera collected at different time points.

FIG. 17. Protection conferred by the rP18tri-based vaccine expressingboth influenza viral HA and NP. A. Stained lung sections from animalsvaccinated with either a prime-boost of rP18tri-GFP/GFP, rP18tri-HA/NPor rP18tri-NP/HA. B. Virus titre in lungs. C. Percent weight loss afterPR8 challenge.

FIG. 18. Humoral response in C57BL6 mice and Balb/c mice induced by therP18tri-based vector vaccine expressing HA proteins of both the H1N1 andH3N2 influenza virus subtypes. Mice were inoculated intramuscularlytwice at an interval of 2 weeks between prime and boost, either withrP18tri-GFP/GFP or with 10⁴ pfu of rP18tri-GFP/HA, rP18tri-GFP/HA3 andrP18tri-HA/HA3. Two weeks after boosting, serum samples were collectedand analysed for HI titres against the PR8 (H1N1) and X31 (H3N2)challenged viruses. A represents HI titre after 2 wks of priming and ●represents HI titre after 2 wks of boosting. Left panel: HI titre inC57BL6 mice. Right panel: HI titre in Balb/c mice.

FIG. 19. Protection against dual influenza virus challenge. Mice werevaccinated with 10⁴ pfu/ml rP18tri-HA/HA3. Five or six days after viruschallenge, virus titres in the lungs were determined. A. Graph showsviral titres when challenged with either the PR8 or X31 influenza virus.B. Weight loss after challenge with 10LD₅₀ A/PR/8 or 10⁵ pfu X31. C. H&Estained sections of lungs harvested 6 days after challenge with 10LD₅₀of A/PR/8 virus and 10⁵ pfu of X31.

FIG. 20. rP18tri vector induces stronger CTL responses after boosting

FIG. 21. Recombinant NP RNase mutant viruses induced type-I IFNsproduction in infected cells. (A) Alanine-substitution mutations at eachof the 5 catalytic residues abolished the ability of PICV NP to suppressthe Sendai virus-induced IFNβ activation (Qi et al., 2010, Nature468:779-783). 293T cells were transfected with an IFNβ promoterdirected-LUC plasmid and the β-gal plasmid, together with an emptyvector or the respective NP plasmids, followed by Sendai virusinfection. LUC activity was measured and normalized for transfectionefficiency by β-gal activity. The results shown are the average of threeindependent experiments. Expression of the WT (rP2 and rP18) and mutantPICV NP proteins was detected by Western blotting using anti-mycantibody. (B) Recombinant PICV RNase mutant viruses produced high levelsof type I IFNs upon viral infection. A549 cells were mock infected orinfected with the respective recombinant PICV viruses at MOI of 1.Supernatants were collected at 12 and 24 hpi, UV-treated to inactivateviral particles, and subjected to the rNDV-GFP-based biological assay tomeasure the level of IFNs. Representative GFP images (B) and the averageGFP values with standard deviations from three independent experiments(C) are shown.

FIG. 22. The NP RNase activity is required for PICV replication in theIFN-competent cells in vitro and PICV infection in vivo. (A) Viralgrowth kinetics in the IFN-defective Vero and IFN-competent A549 cells.Cells were infected with the respective viruses at MOI of 0.01. Atdifferent times post infection, virus titers in the supernatants werequantified by plaque assay. The results shown are the average of threeindependent experiments. (B) Survival rate (left panel) and thenormalized body weight (right panel) of guinea pigs (n=6) infected withthe respective recombinant PICV viruses. Statistical analyses of thesurvival curves were performed using the Log-rank (Mantel-Cox) χ² Testusing GraphPad prism 5 software. ***, p<0.001. **, p<0.01. Body weightof each animal was monitored daily and normalized to day 0. The resultsshown are the average of the normalized body weight for each group.

FIG. 23. The PICV NP RNase mutants induced strong IFN responses to blockthe establishment of virus infection. Guinea pigs were infected i.p.with 1×10⁴ pfu of the respective recombinant viruses (n=3 animals pervirus group) for 1 and 3 days. (A) Viral titers in the livers andspleens at 1 and 3 dpi. (B) The levels of type I IFNs and selectiveinterferon-stimulating genes (ISGs) in the peritoneal cavity cells atday 1 were measured by qRT-PCR. Primer sequences will be provided uponrequest. Each dot represents a single animal. Statistical analyses wereconducted by the student's t test.

FIG. 24. Generation of rP18tri expressing dual antigens flu HA and NP.(A) Schematic diagram of rP18tri vectors expressing dual antigens HA andNP of influenza A virus (IAV) A/PR8 on S1 and S2 segments, rP18tri-P/H,and rP18tri-H/P. IGR, intergenic region. (B) The rP18tri dual antigenvectors express both HA and NP as shown by immunofluorescence assay(IFA). (C) Growth curve analysis of the recombinant viruses expressingthe dual antigens in BHK-21 cells.

FIG. 25. The HA/NP dual antigen expressing vaccine vectors can induceprotective immunity in mice. A group of C57BL6 mice (n =3) were giventwo doses of respective rP18tri vectors at 1×10⁴ pfu and challengedintranasally (IN) with 10×MLD₅₀ (median lethal dose) of themouse-adapted influenza A/PR8 virus. Body weight (A) and viral lungtiters at 3 and 6 dpi (B) are shown.

FIG. 26. The H1/H3 dual antigen vector induces balanced HA neutralizingantibodies. (A) Schematic diagram of rP18tri vectors expressing eGFP andH1 HA (rP18tri-G/H1), eGFP and H3 HA (rP18tri-G/H3), and H1 and H3(rP18tri-H3/H1). (B) The rP18tri-H3/H1 induces balanced neutralizingantibodies against both H1 and H3 HA subtypes. Groups of C57BL6 (toppanels) and Balb/c mice (bottom panels) were immunized with therespective rP18tri vectors (n>=3) twice through the IM route. Bloodcollected at 14 days post prime and post boost were quantified for thelevels of neutralizing antibodies against A/PR8 (H1N1) and A/x31 (H3N2)by HAI assay.

FIG. 27. Induction of heterosubtypic neutralizing antibodies by aprime-and-boost strategy with different HA subtypes. (A) Schematicdiagram of rP18tri vectors expressing A/PR8 NP together with either H1HA from A/PR8 (rP18tri-P/H1) or H3 HA from A/x31 (rP18tri-P/H3). (B)NP-specific CTLs increased upon a booster dose. C57BL6 mice were primedwith rP18tri-P/H1, boosted with rP18tri-P/H3, and boosted again withrP18tri-P/H1, at a 14-day interval. NP-specific effector T cells 7 daysafter prime (dpp), after the 1^(st) boost (dpb), and after the 2^(nd)boost (dpb2) were quantified by the established NP tetramer analysis.(C) Neutralizing antibodies against A/PR8 (H1), A/x31 (H3), and A/WSN(heterosubtypic H1) after prime and boosts were quantified by HAI assay.

FIG. 28. The rP18tri vector can induce both humoral and T cell responsesthrough oral route. Groups of BL6 mice were immunized twice with eitherrP18tri-G (n=3) vector or rP18tri-P/H (n=4) through oral gavage.NP-specific effector T cells (A) and H1-specific neutralizing antibodies(B) at different days after prime or boost are shown. dpp, dayspost-prime; dpb, days post-boost.

FIG. 29. Inactivated rP18tri vector can induce both humoral and T cellresponses. Groups of C57BL6 mice were immunized twice with rP18tri-Gvector (n=3), hydrogen peroxide-inactivated rP18tri-P/H (n=3), and liverP18tri-P/H (n=1), respectively. NP-specific effector T cells (leftpanel) and neutralization antibodies (right) at different days afterimmunization are shown. dpp, days post-prime; dpb, days post-boost.

FIG. 30. The rP18tri vector does not show virulence in guinea pigs andcan protect the animals from a lethal challenge with the WT rP18 virus.(A) The rP18tri-G does not cause virulent infections in guinea pigs.Groups of Hartley guinea pigs (n=3) were mock infected (PBS) or infectedwith WT rP18 (1×10⁴ pfu) or rP18tri-G (1×10⁶ pfu) through the IP route.Body weight was monitored daily and normalized to day 0 (left panel).Rectal temperature is shown on the right. (B) The rP18tri-G-immunizedguinea pigs were protected from lethal rP18 challenge. Groups of guineapigs (n=3) were immunized with either PBS or rP18tri-G at 1×10⁴ pfuthrough the IP route and, 14 days later, challenged with 1×10⁴ pfu of WTrP18 virus. Normalized body weight (left panel) and rectal temperature(right panel) are shown. Temperature above 39.5° C. is considered to befeverish.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

Provided herein is a reverse genetics system for producing geneticallymodified Pichinde virus. The genetically modified Pichinde virus-basedreverse genetics system described herein has multiple advantages overother arenavirus systems. Pichinde virus is not known to cause diseasein humans, and there is evidence that Pichinde virus can causeasymptomatic human infections in a laboratory setting. For instance, 46%of laboratory personnel working with the virus are serum positive but donot show a distinct illness (Buchmeier et al., 2007, Arenaviridae: theviruses and their replication. In: Knipe and Howley (eds), FieldsVirology. 5th ed. Philadelphia, Pa.: Lippincott Williams & Wilkins. pp.1791-1827). The modified Pichinde virus described herein is furtherattenuated, compared to the parental virus used in the human-infectionstudy reported by Buchmeier et al. The modified Pichinde virus isgenetically stable through serial passages in cell cultures. Generalhuman populations are not known to have prior exposure to Pichindevirus, which makes it an ideal vector for vaccine development due to thelack of pre-existing immunity against this Pichinde virus vector. Asused herein, “genetically modified” and “genetically engineered” refersto a Pichinde virus which has been modified and is not found in anynatural setting. For example, a genetically modified Pichinde virus isone into which has been introduced an exogenous polynucleotide, such asa restriction endonuclease site. Another example of a geneticallymodified Pichinde virus is one which has been modified to include threegenomic segments.

The reverse genetics system for this modified Pichinde virus includestwo to three genomic segments. The first genomic segment includes twocoding regions, one that encodes a Z protein and a second that encodes aRNA-dependent RNA polymerase (L RdRp). The second genomic segmentincludes a coding region that encodes a nucleoprotein (NP), and mayinclude at least one restriction enzyme site, such as a multiple cloningsite. The third genomic segment includes a coding region that encodes aglycoprotein, and may include at least one restriction enzyme site, suchas a multiple cloning site. A “coding region” is a nucleotide sequencethat encodes a protein and, when placed under the control of appropriateregulatory sequences expresses the encoded protein. As used herein, theterm “protein” refers broadly to a polymer of two or more amino acidsjoined together by peptide bonds. The term “protein” also includesmolecules which contain more than one protein joined by disulfide bonds,ionic bonds, or hydrophobic interactions, or complexes of proteins thatare joined together, covalently or noncovalently, as multimers (e.g.,dimers, tetramers). Thus, the terms peptide, oligopeptide, andpolypeptide are all included within the definition of protein and theseterms are used interchangeably. It should be understood that these termsdo not connote a specific length of a polymer of amino acids, nor arethey intended to imply or distinguish whether the protein is producedusing recombinant techniques, chemical or enzymatic synthesis, or isnaturally occurring.

The Z protein, L RdRp, NP protein, and glycoprotein are those encoded bya Pichinde virus. The Z protein is a small RING-domain containing matrixprotein that mediates virus budding and also regulates viral RNAsynthesis. One example of a Z protein from a Pichinde virus is thesequence available at Genbank accession number ABU39910.1 (SEQ ID NO:1).The L RdRp protein is a RNA-dependent RNA polymerase that is requiredfor viral DNA synthesis. One example of a L RdRp protein from a Pichindevirus is the sequence available at Genbank accession number ABU39911.1(SEQ ID NO:2). The NP protein encapsidates viral genomic RNAs, isrequired for viral RNA synthesis, and also suppresses host innate immuneresponses. One example of a NP protein from a Pichinde virus is thesequence available at Genbank accession number ABU39909.1 (SEQ ID NO:3).The glycoprotein is post-translationally processed into a stable signalpeptide (SSP), the receptor-binding G1 protein, and the transmembrane G2protein. One example of a glycoprotein from a Pichinde virus is thesequence available at Genbank accession number ABU39908.1 (SEQ ID NO:4).

Other examples of Z proteins, L RdRp proteins, NP proteins, andglycoprotein include proteins having structural similarity with aprotein that is encoded by a Pichinde virus, for instance, SEQ ID NO:1,2, 3, and/or 4. Structural similarity of two polypeptides can bedetermined by aligning the residues of the two polypeptides (forexample, a candidate polypeptide and a reference polypeptide describedherein) to optimize the number of identical amino acids along thelengths of their sequences; gaps in either or both sequences arepermitted in making the alignment in order to optimize the number ofidentical amino acids, although the amino acids in each sequence mustnonetheless remain in their proper order. A reference polypeptide may bea polypeptide described herein, such as SEQ ID NO:1, 2, 3, or 4. Acandidate polypeptide is the polypeptide being compared to the referencepolypeptide. A candidate polypeptide may be isolated, for example, froma cell of an animal, such as a mouse, or can be produced usingrecombinant techniques, or chemically or enzymatically synthesized. Acandidate polypeptide may be inferred from a nucleotide sequence presentin the genome of a Pichinde virus.

Unless modified as otherwise described herein, a pair-wise comparisonanalysis of amino acid sequences can be carried out using the Blastpprogram of the blastp suite-2sequences search algorithm, as described byTatiana et al., (FEMS Microbiol Lett, 174, 247-250 (1999)), andavailable on the National Center for Biotechnology Information (NCBI)website. The default values for all blastp suite-2sequences searchparameters may be used, including general parameters: expectthreshold=10, word size=3, short queries=on; scoring parameters:matrix=BLOSUM62, gap costs=existence:11 extension:1, compositionaladjustments=conditional compositional score matrix adjustment.Alternatively, polypeptides may be compared using the BESTFIT algorithmin the GCG package (version 10.2, Madison Wis.).

In the comparison of two amino acid sequences, structural similarity maybe referred to by percent “identity” or may be referred to by percent“similarity.” “Identity” refers to the presence of identical aminoacids. “Similarity” refers to the presence of not only identical aminoacids but also the presence of conservative substitutions. Aconservative substitution for an amino acid in a polypeptide describedherein may be selected from other members of the class to which theamino acid belongs. For example, it is known in the art of proteinbiochemistry that an amino acid belonging to a grouping of amino acidshaving a particular size or characteristic (such as charge,hydrophobicity and hydrophilicity) can be substituted for another aminoacid without altering the activity of a protein, particularly in regionsof the protein that are not directly associated with biologicalactivity. For example, nonpolar (hydrophobic) amino acids includealanine, leucine, isoleucine, valine, proline, phenylalanine,tryptophan, and tyrosine. Polar neutral amino acids include glycine,serine, threonine, cysteine, tyrosine, asparagine and glutamine. Thepositively charged (basic) amino acids include arginine, lysine andhistidine. The negatively charged (acidic) amino acids include asparticacid and glutamic acid. Conservative substitutions include, for example,Lys for Arg and vice versa to maintain a positive charge; Glu for Aspand vice versa to maintain a negative charge; Ser for Thr so that a free—OH is maintained; and Gln for Asn to maintain a free —NH2.

The skilled person will recognize that the Z protein depicted at SEQ IDNO:1 can be compared to Z proteins from other arenaviruses, includingLassa virus (O73557.4), LCMV Armstrong (AAX49343.1), and Junin virus(NP_899216.1) using readily available algorithms such as ClustalW toidentify conserved regions of Z proteins. ClustalW is a multiplesequence alignment program for nucleic acids or proteins that producesbiologically meaningful multiple sequence alignments of differentsequences (Larkin et al., 2007, ClustalW and ClustalX version 2,Bioinformatics, 23(21):2947-2948). Using this information the skilledperson will be able to readily predict with a reasonable expectationthat certain conservative substitutions to an Z protein such as SEQ IDNO:1 will not decrease activity of the polypeptide.

The skilled person will recognize that the L RdRp protein depicted atSEQ ID NO:2 can be compared to L RdRp proteins from other arenaviruses,including Lassa virus (AAT49002.1), LCMV Armstrong (AAX49344.1), andJunin virus (NP 899217.1) using readily available algorithms such asClustalW to identify conserved regions of L RdRp proteins. Using thisinformation the skilled person will be able to readily predict with areasonable expectation that certain conservative substitutions to an LRdRp protein such as SEQ ID NO:2 will not decrease activity of thepolypeptide.

The skilled person will recognize that the NP protein depicted at SEQ IDNO:3 can be compared to NP proteins from other arenaviruses, includingLassa virus (P13699.1), LCMV Armstrong (AAX49342.1), and Junin virus (NP899219.1) using readily available algorithms such as ClustalW toidentify conserved regions of NP proteins. Using this information theskilled person will be able to readily predict with a reasonableexpectation that certain conservative substitutions to a NP protein suchas SEQ ID NO:3 will not decrease activity of the polypeptide.

The skilled person will recognize that the glycoprotein depicted at SEQID NO:4 can be compared to glycoproteins from other arenaviruses,including Lassa virus (P08669), LCMV Armstrong (AAX49341.1), and Juninvirus (NP_899218.1) using readily available algorithms such as ClustalWto identify conserved regions of glycoproteins. Using this informationthe skilled person will be able to readily predict with a reasonableexpectation that certain conservative substitutions to a glycoproteinsuch as SEQ ID NO:4 will not decrease activity of the polypeptide.

Thus, as used herein, a Pichinde virus Z protein, L RdRp protein, an NPprotein, or a glycoprotein includes those with at least 50%, at least55%, at least 60%, at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 86%, at least 87%, at least 88%, at least89%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least99% amino acid sequence similarity to a reference amino acid sequence.Alternatively, as used herein, a Pichinde virus Z protein, L RdRpprotein, an NP protein, or a glycoprotein includes those with at least50%, at least 55%, at least 60%, at least 65%, at least 70%, at least75%, at least 80%, at least 85%, at least 86%, at least 87%, at least88%, at least 89%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, or at least 99% amino acid sequence identity to a reference aminoacid sequence. Unless noted otherwise, “Pichinde virus Z protein,”“Pichinde virus L RdRp protein,” “Pichinde virus NP protein,” and“Pichinde virus glycoprotein” refer to a protein having at least 80%amino acid identity to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ IDNO:4, respectively.

A Pichinde virus Z protein, L RdRp protein, an NP protein, or aglycoprotein having structural similarity the amino acid sequence of SEQID NO:1, 2, 3, or 4, respectively, has biological activity. As usedherein, “biological activity” refers to the activity of Z protein, LRdRp protein, an NP protein, or a glycoprotein in producing aninfectious virus particle. The biological role each of these proteinsplay in the biogenesis of an infectious virus particle is knows, as areassays for measuring biological activity of each protein.

In one embodiment, the NP protein may include one or more mutations. Amutation in the NP protein may result in a NP protein that continues tofunction in the production of infectious viral particles, but has adecreased ability to suppress the production of certain cytokines by acell infected with a Pichinde virus. A Pichinde virus that has decreasedability to suppress cytokine production is expected to be useful inenhancing an immunological response to an antigen encoded by the virus.Examples of mutations include the aspartic acid at residue 380, theglutamic acid at residue 382, the aspartic acid at residue 457, theaspartic acid at residue 525, and the histidine at residue 520. A personof ordinary skill in the art recognizes that the precise location ofthese mutations can vary between different NP proteins depending uponthe presence of small insertions or deletions in the NP protein, thusthe precise location of a mutation is approximate, and can vary by 1, 2,3, 4, or 5 amino acids.

In one embodiment, the mutation in the NP protein may be the replacementof the aspartic acid, glutamic acid, or histidine at residues 380, 382,457, 525, and/or 520 with any other amino acid. In one embodiment, themutation may be the conservative substitution of the aspartic acid,glutamic acid, or histidine at residues 380, 382, 457, 525, and/or 520.In one embodiment, the mutation may be the replacement of the asparticacid, glutamic acid, or histidine at residues 380, 382, 457, 525, and/or520 with a glycine or an alanine. In one embodiment, the NP protein mayinclude a mutation at one, two, three, or four of the residues 380, 382,457, 525, or 520, and in one embodiment the NP protein may include amutation at all five residues.

In one embodiment, the glycoprotein may include one or more mutations. Amutation in the glycoprotein may result in a glycoprotein that impairsvirus spreading in vivo. Examples of mutations include the asparagine atresidue 20, and/or the asparagine at residue 404. A person of ordinaryskill in the art recognizes that the precise location of these mutationscan vary between different glycoproteins depending upon the presence ofsmall insertions or deletions in the glycoprotein, thus the preciselocation of a mutation is approximate, and can vary by 1, 2, 3, 4, or 5amino acids.

In one embodiment, the mutation in the glycoprotein may be thereplacement of the asparagine residue 20 and/or 404 with any other aminoacid. In one embodiment, the mutation may be the conservativesubstitution of the asparagine residue 20 and/or 404. In one embodiment,the mutation may be the replacement of the asparagine residue 20 and/or404 with a glycine or an alanine.

Proteins as described herein also may be identified in terms thepolynucleotide that encodes the protein. Thus, this disclosure providespolynucleotides that encode a protein as described herein or hybridize,under standard hybridization conditions, to a polynucleotide thatencodes a protein as described herein, and the complements of suchpolynucleotide sequences. As used herein, the term “polynucleotide”refers to a polymeric form of nucleotides of any length, eitherribonucleotides or deoxynucleotides, and includes both double- andsingle-stranded DNA and RNA. A polynucleotide may include nucleotidesequences having different functions, including for instance codingsequences, and non-coding sequences such as regulatory sequences. Apolynucleotide can be obtained directly from a natural source, or can beprepared with the aid of recombinant, enzymatic, or chemical techniques.A polynucleotide can be linear or circular in topology. A polynucleotidecan be, for example, a portion of a vector, such as an expression orcloning vector, or a fragment. An example of a polynucleotide is agenomic segment.

An example of a polynucleotide encoding a Z protein is the nucleotides85-372 of the sequence available at Genbank accession number EF529747.1(SEQ ID NO:5), an example of a polynucleotide encoding an L RdRp proteinis the complement of nucleotides 443-7027 of the sequence available atGenbank accession number EF529747.1 (SEQ ID NO:5), an example of apolynucleotide encoding an NP protein is the complement of nucleotides1653..3338 of the sequence available at Genbank accession numberEF529746.1 (SEQ ID NO:6), and an example of a polynucleotide encoding aglycoprotein protein is the nucleotides 52-1578 of the sequenceavailable at Genbank accession number EF529746.1 (SEQ ID NO:6). Itshould be understood that a polynucleotide encoding a Z protein, an LRdRp protein, an NP protein, or a glycoprotein represented by SEQ IDNO:1, 2, 3, or 4, respectively, is not limited to the nucleotidesequence disclosed at SEQ ID NO:5 or 6, but also includes the class ofpolynucleotides encoding such proteins as a result of the degeneracy ofthe genetic code. For example, the naturally occurring nucleotidesequence SEQ ID NO:5 is but one member of the class of nucleotidesequences encoding a protein having the amino acid sequence SEQ ID NO:1and a protein having the amino acid sequence SEQ ID NO:2. The class ofnucleotide sequences encoding a selected protein sequence is large butfinite, and the nucleotide sequence of each member of the class can bereadily determined by one skilled in the art by reference to thestandard genetic code, wherein different nucleotide triplets (codons)are known to encode the same amino acid.

As used herein, reference to a polynucleotide as described herein and/orreference to the nucleic acid sequence of one or more SEQ ID NOs caninclude polynucleotides having a sequence identity of at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 86%, at least 87%, at least 88%, at least 89%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% sequence identityto an identified reference polynucleotide sequence.

In this context, “sequence identity” refers to the identity between twopolynucleotide sequences. Sequence identity is generally determined byaligning the bases of the two polynucleotides (for example, aligning thenucleotide sequence of the candidate sequence and a nucleotide sequencethat includes, for example, a nucleotide sequence that encodes a proteinof SEQ ID NO:1, 2, 3, or 4) to optimize the number of identicalnucleotides along the lengths of their sequences; gaps in either or bothsequences are permitted in making the alignment in order to optimize thenumber of shared nucleotides, although the nucleotides in each sequencemust nonetheless remain in their proper order. A candidate sequence isthe sequence being compared to a known sequence—e.g., a nucleotidesequence that includes the appropriate nucleotide sequence selectedfrom, for example, SEQ ID NO:5 or 6. For example, two polynucleotidesequences can be compared using the Blastn program of the BLAST 2 searchalgorithm, as described by Tatiana et al., FEMS Microbiol Lett.,1999;174: 247-250, and available on the world wide web atncbi.nlm.nih.gov/BLAST/. The default values for all BLAST 2 searchparameters may be used, including reward for match=1, penalty formismatch=−2, open gap penalty=5, extension gap penalty=2, gapx_dropoff=50, expect=10, wordsize=11, and filter on.

In one embodiment, the second and/or third genomic segments may eachindependently include a “multiple cloning site” with one restrictionsite or more than one restriction site.

In one embodiment, the second and/or third genomic segments may eachindependently include an additional coding region that encodes aprotein, such as an antigen. Thus, the second genomic segment includesthe coding region encoding the nucleoprotein and may include a secondcoding region that encodes an antigen. Likewise, the third genomicsegment includes the coding region encoding the glycoprotein and mayinclude a second coding region that encodes an antigen. The second andthird genomic segments may encode the same antigen or differentantigens. In both the second genomic segment and the third genomicsegment this second coding region may be inserted into a restrictionsite present, such as a restriction site present in a multiple cloningsite. The second coding region that may be present on the second genomicsegment and/or the third genomic segment is not intended to be limiting.

In one embodiment, the second coding region may encode a protein that isuseful as an antigen that can elicit an immune response in a subject.Examples 1, 2, and 3 show the use of influenza nucleoproteins andinfluenza hemagglutinins as model antigens to demonstrate theeffectiveness of the reverse genetics systems and viruses disclosedherein, and accordingly the identity of the antigen is not intended tobe limiting. An example of an antigen is a full length protein encodedby a prokaryotic cell, a eukaryotic cell (including, for instance afungus, yeast, or protozoan) or a virus, or a fragment thereof. In oneembodiment, the protein may be one that is naturally expressed by apathogen, such as a viral pathogen, a prokaryotic pathogen, or aeukaryotic pathogen. Antigenic proteins encoded by a prokaryotic cell, aeukaryotic cell, or a virus are known to the skilled person in the art.Another example of an antigen is a protein engineered to include one ormore epitopes. In one embodiment, the protein may be one that isexpressed by a tumor cell or is present in a tumor environment.

Examples of prokaryotic pathogens from which an antigen may be obtainedinclude gram negative pathogens and gram positive pathogens. Examples ofgram negative pathogens include enteropathogens, such as members of thefamily Enterobacteriaceae, including members of the familyEnterobacteriaceae that are members of the tribe Escherichieae orSalmonelleae. Examples of enteropathogens include members of the familyEnterobacteriaceae, members of the family Vibrionaceae (including, forinstance, Vibrio cholerae), and Campylobacter spp. (including, forinstance, C. jejuni). Examples of preferred members of the familyEnterobacteriaceae include, for instance, E. coli, Shigella spp.,Salmonella spp., Proteus spp., Klebsiella spp. (for instance, K.pneumoniae), Serratia spp., and Yersinia spp. Examples of strains of E.coli include, for example, E. coli scrotypes O1a, O2a, O78, and O157,different O:H serotypes including 0104, 0111, 026, 0113, 091, hemolyticstrains of enterotoxigenic E. coli such as K88+, F4+, F18ab+, andF18ac+, and non-hemolytic strains such as 987P, F41, and K99. As usedherein, the term “strain” refers to members of a species of microbewhere the members have different genotypes and/or phenotypes. Otherexamples of E. coli include five categories of diarrheagenic Escherichiacoli that cause foodborne and waterborne diseases in humans: theenteropathogenic (EPEC), enterohemorrhagic (EHEC), enterotoxigenic(ETEC), enteroinvasive (EIEC) and enteroaggregative (EAEC) strains.Other gram negative microbes include members of the familyPasteurellaceae, preferably Pasturella spp., such as P. multocida, andMannheimia haemolytica, and members of the family Pseudomonadaceae, suchas Pseudomonas spp., including P. aeruginosa. Yet other gram negativemicrobes include Bordetella spp., Burkholderia spp., Chlamydia spp.,Enterobacter spp., Helobacter spp., Histophilus spp., Moraxella spp.,Legionella spp., Leptospiria spp., Rickettsia spp., Treponnema spp.,Neisseria spp., Actinobacillus spp., Haemophilus spp., Myxcobacteriaspp., Sporocytophaga spp., Chondrococcus spp., Cytophaga spp.,Flexibacter spp., Flavobacterium spp., Aeromonas spp. Examples ofYersinia spp. include Y. pestis, Y. pseudotuberculosis, and Y.enterocolitica. Examples of Fusobacterium spp. belonging to the familyFusobacteriaceae include F. necrophorum (including subspecies F.necrophorum subsp. necrophorum and F. necrophorum subsp. funduliforme),F. nucleatum, F. canifelinum, F. gonidiaformans, F. mortiferum, F.naviforme, F. necrogenes, F. russii, F. ulcerans, and F. variu.

Examples of gram-positive pathogens include members of the familyMicrococcaceae, including Staphylococcus spp., such as S. aureus. Othergram positive pathogens include members of the family Deinococcaceae,such as Streptococcus agalactiae, Streptococcus uberis, Streptococcusbovis, Streptococcus equi, Streptococcus zooepidemicus, andStreptococcus dysgalatiae. Other gram positive microbes from which anantigen may be obtained include Bacillus spp., Clostridium spp.,Corynebacterium spp., Erysipelothrix spp., Listeria spp., Mycobacteriumspp., Other gram positive pathogens include members of theCorynebacteriaceae, Enterococcacae, Micrococcaceae, Mycobacteriaceae,Nocardiaceae, and Peptococcaceae, which include such bacterial speciesas Actinomyces spp., Bifidobacterium spp., Enterococcus spp.,Eubacterium spp., Kytococcus spp., Lactobacillus spp., Micrococcus spp.,Mobiluncus spp., Mycobacteria spp., Peptostreptococcus spp., andPropionibacterium spp., Gardnerella spp., Mycoplasma, Norcardia spp.,Streptomyces spp., Borrelia spp., and Bacillus spp.

Examples of pathogenic protozoa include intestinal protozoa, urogenitalprotozoa, Systemic protozoa, and extraintestinal protozoa. Specificexamples include, but are not limited to, e.g. Entamoeba histolytica,Giardia lamblia, Cryptosporidium parvum, Cystoisospora belli, Cyclosporacayetanensis, members of the phylum Microsporidia, Trichomonasvaginalis, Plamodium falciparum, Toxoplasma gondii, members of thesubclass Coccidiosis such as members of the genus Eimeria, including E.acervulina., E. necatrix and E. tenella, Nematodes, Trematodes, andCestodes.

Examples of viral pathogens include, but are not limited to members ofthe family Rhabdoviridae (including Vesicular stomatitis virus VSV),members of the genus Aphthovirus (including Foot-and-mouth disease virusFMDV), members of the genus Pestivirus (including Bovine viral diarrheavirus), members of the family Arterivirus (including porcinereproductive and respiratory syndrome virus PRRSV), Coronaviruses(including Porcine Epidemic Diarrhea virus EPDV, SARS-CoV, MERS-CoV),members of the genus Torovirus (including Equine torovirus), members ofthe family Orthomyxoviridae (including influenza virus), members of thefamily Reoviridae (including rotavirus, Bluetongue disease virus, avianreoviruses), members of the family Circovirus, members of the familyHerpesviridae, members of the family Retroviridae (including HIV, FIV,SIV, ALV, BLV, RSV), members of the Asfariridae family (includingAfrican swine fever virus), members of the genus Flavivirus (includingDengue virus, Yellow fever virus), members of the family Paramyxoviridae(including Newcastle disease virus and Respiratory syncytial virus RSV),as well as members of the genus arenavirus (including members of theLCMV-Lassa virus (Old World) complex and the Tacaribe virus (New World)complex). Specific non-limiting examples of members of the genusarenavirus include Lymphocytic choriomeningitis virus, Pichinde virus,Lassa virus, Mopeia virus, Junin virus, Guanarito virus, Lujo virus ,Machupo virus, Sabia virus, and Whitewater Arroyo virus.

In one embodiment, the protein encoded by the second coding region maybe from an influenza virus, such as a nucleoprotein, a hemagglutinin, ora portion thereof. The nucleoprotein may be of any subtype, includingbut not limited to, A/PR8 NP (e.g., Genbank accession numberNP_040982.1). The hemagglutinin may be of any subtype, including but notlimited to, H1 and H3.

The protein encoded by the second coding region may be one that resultsin a humoral immune response, a cell-mediated immune response, or acombination thereof. In one embodiment, the protein encoded by thesecond coding region of the second and third genomic segments is atleast 6 amino acids in length. The antigen may be heterologous to thecell in which the coding region is expressed. The nucleotide sequence ofa second coding region present on a second and third genomic segment andencoding the antigen can be readily determined by one skilled in the artby reference to the standard genetic code. The nucleotide sequence of asecond coding region present on a second and third genomic segment andencoding the antigen may be modified to reflect the codon usage bias ofa cell in which the antigen will be expressed. The usage bias of nearlyall cells in which a Pichinde virus would be expressed is known to theskilled person.

In one embodiment, the second coding region may encode a protein that isuseful as a detectable marker, e.g., a molecule that is easily detectedby various methods. Examples include fluorescent polypeptides (e.g.,green, yellow, blue, or red fluorescent proteins), luciferase,chloramphenicol acetyl transferase, and other molecules (such as c-myc,flag, 6×his, HisGln (HQ) metal-binding peptide, and V5 epitope)detectable by their fluorescence, enzymatic activity or immunologicalproperties.

When the second and/or third genomic segments include a coding regionthat encodes an antigen, the maximum size in nucleotides of the codingregion(s) is determined by considering the total size of the secondgenomic segment and the third genomic segment. The total size of the twogenomic segments may be no greater than 3.4 kilobases (kb), no greaterthan 3.5 kb, no greater than 3.6 kb, no greater than 3.7 kb, no greaterthan 3.8 kb, no greater than 3.9 kb, no greater than 4.0 kb, no greaterthan 4.1 kb, no greater than 4.2 kb, no greater than 4.3 kb, no greaterthan 4.4 kb, or no greater than 4.5 kb.

Pichinde virus is an arenavirus, and one characteristic of an arenavirusis an ambisense genome. As used herein, “ambisense” refers to a genomicsegment having both positive sense and negative sense portions. Forexample, the first genomic segment of a Pichinde virus described hereinis ambisense, encoding a Z protein in the positive sense and encoding aL RdRp protein in the negative sense. Thus, one of the two codingregions of the first genomic segment is in a positive-sense orientationand the other is in a negative-sense orientation. When the second and/orthe third genomic segment includes a second coding region encoding anantigen, the coding region encoding the antigen is in a negative-senseorientation compared to the NP protein of the second genomic segment andto the glycoprotein of the third genomic segment.

Each genomic segment also includes nucleotides encoding a 5′untranslated region (UTR) and a 3′ UTR. These UTRs are located at theends of each genomic segment. Nucleotides useful as 5′ UTRs and 3′ UTRsare those present in Pichinde virus and are readily available to theskilled person (see, for instance, Buchmeier et al., 2007, Arenaviridae:the viruses and their replication. In: Knipe and Howley (eds), FieldsVirology. 5th ed. Philadelphia, Pa.: Lippincott Williams & Wilkins. pp.1791-1827). In one embodiment, a genomic segment that encodes a Zprotein and an L RdRp protein includes a 5′ UTR sequence that is 5′CGCACCGGGGAUCCUAGGCAUCUUUGGGUCACGCUUCAAAUUUGUCCAAUUUGAACCCAGCUCAAGUCCUGGUCAAAACUUGGG (SEQ ID NO:8) and a 3′ UTR sequence thatis CGCACCGAGGAUCCUAGGCAUUUCUUGAUC (SEQ ID NO:9). In one embodiment, agenomic segment that encodes a NP protein or a glycoprotein includes a5′ UTR sequence that is 5′CGCACCGGGGAUCCUAGGCAUACCUUGGACGCGCAUAUUACUUGAUCAAAG (SEQ ID NO:10) and a3′ UTR sequence that is 5′CGCACAGUGGAUCCUAGGCGAUUCUAGAUCACGCUGUACGUUCACUUCUUCACUGACUCGGAGGAAGUGCAAACAACCCCAAA (SEQ ID NO:11). Alterations in thesesequences are permitted, and the terminal 27-30 nucleotides are highlyconserved between the genomic segments.

Each genomic segment also includes an intergenic region located betweenthe coding region encoding a Z protein and the coding region encoding aL RdRp protein, between the coding region encoding a nucleoprotein andthe at least one first restriction enzyme site, and between the codingregion encoding a glycoprotein and at least one second restrictionenzyme site. Nucleotides useful as an intergenic region are thosepresent in Pichinde virus and are readily available to the skilledperson. In one embodiment, an IGR sequence of a genomic segment thatencodes a Z protein and an L RdRp protein includes 5′ACCAGGGCCCCUGGGCGCACCCCCCUCCGGGGGUGCGCCCGGGGGCCCCCGGCCCC AUGGGGCCGGUUGUU(SEQ ID NO:12). In one embodiment, an IGR sequence of a genomic segmentthat encodes a NP protein or a glycoprotein includes 5′GCCCUAGCCUCGACAUGGGCCUCGACGUCACUCCCCAAUAGGGGAGUGACGUCGAGGCCUCUGAGGACUUGAGCU (SEQ ID NO:13).

In one embodiment, each genomic segment is present in a vector. In oneembodiment, the sequence of a genomic segment in the vector isantigenomic, and in one embodiment the sequence of a genomic segment inthe vector is genomic. As used herein, “anti-genomic” refers to agenomic segment that encodes a protein in the orientation opposite tothe viral genome. For example, Pichinde virus is a negative-sense RNAvirus. However, each genomic segment is ambisense, encoding proteins inboth the positive-sense and negative-sense orientations. “Anti-genomic”refers to the positive-sense orientation, while “genomic” refers to thenegative-sense orientation.

A vector is a replicating polynucleotide, such as a plasmid, phage, orcosmid, to which another polynucleotide may be attached so as to bringabout the replication of the attached polynucleotide. Construction ofvectors containing a genomic segment, and construction of genomicsegments including insertion of a polynucleotide encoding an antigen,employs standard ligation techniques known in the art. See, e.g.,Sambrook et al, Molecular Cloning: A Laboratory Manual., Cold SpringHarbor Laboratory Press (1989) or Ausubel, R. M., ed. Current Protocolsin Molecular Biology (1994). A vector can provide for further cloning(amplification of the polynucleotide), i.e., a cloning vector, or forexpression of an RNA encoded by the genomic segment, i.e., an expressionvector. The term vector includes, but is not limited to, plasmidvectors, viral vectors, cosmid vectors, or artificial chromosomevectors. Typically, a vector is capable of replication in a prokaryoticcell and/or a eukaryotic cell. In one embodiment, the vector replicatesin prokaryotic cells, and not in eukaryotic cells. In one embodiment,the vector is a plasmid.

Selection of a vector depends upon a variety of desired characteristicsin the resulting construct, such as a selection marker, vectorreplication rate, and the like. Suitable host cells for cloning orexpressing the vectors herein are prokaryote or eukaryotic cells.

An expression vector optionally includes regulatory sequences operablylinked to the genomic segment. The term “operably linked” refers to ajuxtaposition of components such that they are in a relationshippermitting them to function in their intended manner. A regulatorysequence is “operably linked” to a genomic segment when it is joined insuch a way that expression of the genomic segment is achieved underconditions compatible with the regulatory sequence. One regulatorysequence is a promoter, which acts as a regulatory signal that bind RNApolymerase to initiate transcription of the downstream (3′ direction)genomic segment. The promoter used can be a constitutive or an induciblepromoter. The invention is not limited by the use of any particularpromoter, and a wide variety of promoters are known. In one embodiment,a T7 promoter is used. Another regulatory sequence is a transcriptionterminator located downstream of the genomic segment. Any transcriptionterminator that acts to stop transcription of the RNA polymerase thatinitiates transcription at the promoter may be used. In one embodiment,when the promoter is a T7 promoter, a T7 transcription terminator isalso used. In one embodiment, a ribozyme is present to aid in processingan RNA molecule. A ribozyme may be present after the sequences encodingthe genomic segment and before a transcription terminator. An example ofa ribozyme is a hepatitis delta virus ribozyme. One example of ahepatitis delta virus ribozyme is 5′AGCTCTCCCTTAGCCATCCGAGTGGACGACGTCCTCCTTCGGATGCCCAGGTCGGACCGCGAGGAGGTGGAGATGCCATGCCGACCC (SEQ ID NO:14).

Transcription of a genomic segment present in a vector results in an RNAmolecule. When each of the three genomic segments is present in a cellthe coding regions of the genomic segments are expressed and viralparticles that contain one copy of each of the genomic segments areproduced. The three genomic segments of the reverse genetics systemdescribed herein are based on Pichinde virus, an arenavirus with asegmented genome of two single-stranded ambisense RNAs. While theability of the reverse genetics system to replicate and produceinfectious virus typically requires the presence of the ambisense RNAsin a cell, the genomic segments described herein also include thecomplement thereof (i.e., complementary RNA), and the corresponding DNAsequences of the two RNA sequences.

The polynucleotide used to transform a host cell optionally includes oneor more marker sequences, which typically encode a molecule thatinactivates or otherwise detects or is detected by a compound in thegrowth medium. For example, the inclusion of a marker sequence canrender the transformed cell resistant to an antibiotic, or it can confercompound-specific metabolism on the transformed cell. Examples of amarker sequence are sequences that confer resistance to kanamycin,ampicillin, chloramphenicol, tetracycline, and neomycin.

Also provided herein are compositions including a viral particledescribed herein, or the three genomic segments described herein. Suchcompositions typically include a pharmaceutically acceptable carrier. Asused herein “pharmaceutically acceptable carrier” includes saline,solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like,compatible with pharmaceutical administration. Additional activecompounds can also be incorporated into the compositions.

A composition described herein may be referred to as a vaccine. The term“vaccine” as used herein refers to a composition that, uponadministration to an animal, will increase the likelihood the recipientmounts an immune response to an antigen encoded by one of the genomicsegments described herein.

A composition may be prepared by methods well known in the art ofpharmaceutics. In general, a composition can be formulated to becompatible with its intended route of administration. Administration maybe systemic or local. Examples of routes of administration includeparenteral (e.g., intravenous, intradermal, subcutaneous,intraperitoneal, intramuscular), enteral (e.g., oral), and topical(e.g., epicutaneous, inhalational, transmucosal) administration.Appropriate dosage forms for enteral administration of the compound ofthe present invention may include tablets, capsules or liquids.Appropriate dosage forms for parenteral administration may includeintravenous administration. Appropriate dosage forms for topicaladministration may include nasal sprays, metered dose inhalers,dry-powder inhalers or by nebulization.

Solutions or suspensions can include the following components: a sterilediluent such as water for administration, saline solution, fixed oils,polyethylene glycols, glycerin, propylene glycol or other syntheticsolvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfate;chelating agents such as ethylenediaminetetraacetic acid; buffers suchas acetates, citrates or phosphates; electrolytes, such as sodium ion,chloride ion, potassium ion, calcium ion, and magnesium ion, and agentsfor the adjustment of tonicity such as sodium chloride or dextrose. pHcan be adjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. A composition can be enclosed in ampoules, disposablesyringes or multiple dose vials made of glass or plastic.

Compositions can include sterile aqueous solutions (where water soluble)or dispersions and sterile powders for the extemporaneous preparation ofsterile solutions or dispersions. For intravenous administration,suitable carriers include physiological saline, bacteriostatic water,phosphate buffered saline (PBS), and the like. A composition istypically sterile and, when suitable for injectable use, should be fluidto the extent that easy syringability exists. It should be stable underthe conditions of manufacture and storage and preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyetheylene glycol, and the like), and suitable mixturesthereof. Prevention of the action of microorganisms can be achieved byvarious antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile solutions can be prepared by incorporating the active compound(e.g., a viral particle described herein) in the required amount in anappropriate solvent with one or a combination of ingredients enumeratedabove, followed by filtered sterilization. Generally, dispersions areprepared by incorporating the active compound into a sterile vehicle,which contains a basic dispersion medium and any other appropriateingredients. In the case of sterile powders for the preparation ofsterile injectable solutions, preferred methods of preparation includevacuum drying and freeze-drying which yields a powder of the activeingredient plus any additional desired ingredient from a previouslysterilized solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules, e.g., gelatin capsules. Oral compositionscan also be prepared using a fluid carrier for use as a mouthwash.Pharmaceutically compatible binding agents, and/or adjuvant materialscan be included as part of the composition. The tablets, pills,capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the active compounds are delivered inthe form of an aerosol spray from a pressured container or dispenserwhich contains a suitable propellant, e.g., a gas such as carbondioxide, or a nebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The active compounds may be prepared with carriers that will protect thecompound against rapid elimination from the body, such as a controlledrelease formulation, including implants. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Suchformulations can be prepared using standard techniques. Liposomalsuspensions can also be used as pharmaceutically acceptable carriers.These can be prepared according to methods known to those skilled in theart.

The data obtained from cell culture assays and animal studies can beused in formulating a range of dosage for use in an animal. The dosageof such compounds lies preferably within a range of circulatingconcentrations that include the ED₅₀ (the dose therapeutically effectivein 50% of the population) with little or no toxicity. The dosage mayvary within this range depending upon the dosage form employed and theroute of administration utilized.

The compositions can be administered once to result in an immuneresponse, or one or more additional times as a booster to potentiate theimmune response and increase the likelihood immunity to the antigen islong-lasting. The skilled artisan will appreciate that certain factorsmay influence the dosage and timing required to effectively treat asubject, including but not limited to the severity of the disease ordisorder, previous treatments, the general health and/or age of thesubject, and other diseases present.

Also provided herein are methods for using the genomic segments. In oneembodiment, a method includes making an infectious viral particle. Sucha method includes, but is not limited to, providing a cell that includeseach of the three genomic segments described herein (a first genomicsegment, a second genomic segment, and a third genomic segment) andincubating the cell under conditions suitable for generating full-lengthgenomic RNA molecules of each genomic segment. The full-length genomicRNA of each genomic segment is antigenomic. Production of full-lengthgenomic RNA molecules of each genomic segment results in transcriptionand translation of each viral gene product and amplification of theviral genome to generate infectious progeny virus particles. As usedherein, an “infectious virus particle” refers to a virus particle thatcan interact with a suitable eukaryotic cell, such as a mammalian cell(e.g., a murine cell or a human cell) or an avian cell, to result in theintroduction of the three genomic segments into the cell, and thetranscription of the three genomic segments in the cell. The method mayalso include introducing into the cell vectors that encode the threegenomic segments. Infectious virus particles are released intosupernatants and may be isolated and amplified further by culturing oncells. The method may include isolating a viral particle from a cell ora mixture of cells and cellular debris. The method may includeinactivating virus particles using standard methods, such a hydrogenperoxide treatment. Also provided is a viral particle, infectious orinactivated, that contains three genomic segments described herein.

In one embodiment, a method includes expression of an antigen in a cell.Such a method includes, but is not limited to, introducing into a cellthe three genomic segments described herein. In one embodiment, theintroducing is by introduction of a virus particle that is infectious orinactivated. The second and/or the third genomic segment may include asecond coding region that encodes an antigen. The second and thirdgenomic segments may encode the same antigen or they may encodedifferent antigens. More than one type of virus particle may beadministered. For instance, two populations of virus particles may beadministered where each population encodes different antigens. In thisembodiment, a single administration can result in expressing multipleantigens in a cell. The cell is a suitable eukaryotic cell, such as amammalian cell (e.g., a murine cell or a human cell) or an avian cell.In one embodiment, the avian cell is a chicken embryonic fibroblast. Thecell may be ex vivo or in vivo. The three genomic segments may beintroduced by contacting a cell with an infectious virus particle thatcontains the three genomic segments, or by introducing into the cellvectors that include the genomic segments. The method further includesincubating the cell under conditions suitable for expression of thecoding regions present on the three genomic segments, including the oneor two second coding regions present on the second and/or third genomicsegments. As used herein, “ex vivo” refers to a cell that has beenremoved from the body of an animal. Ex vivo cells include, for instance,primary cells (e.g., cells that have recently been removed from asubject and are capable of limited growth in tissue culture medium), andcultured cells (e.g., cells that are capable of long term culture intissue culture medium). “In vivo” refers to cells that are within thebody of a subject.

In one embodiment, a method includes immunizing an animal. Such a methodincludes, but is not limited to, administering to an animal a viralparticle that is infectious or inactivated, that contains the threegenomic segments described herein. The second and/or the third genomicsegment may include a second coding region that encodes an antigen. Thesecond and third genomic segments may encode the same antigen or theymay encode different antigens. More than one type of virus particle maybe administered. For instance, two populations of virus particles may beadministered where each population encodes different antigens. In thisembodiment, a single administration can result in vaccinating an animalagainst multiple pathogens. The animal may be any animal in need ofimmunization, including a vertebrate, such as a mammal or an avian. Theanimal can be, for instance, avian (including, for instance, chicken orturkey), bovine (including, for instance, a member of the species Bostaurus), caprine (including, for instance, goat), ovine (including, forinstance, sheep), porcine (including, for instance, swine), bison(including, for instance, buffalo), a companion animal (including, forinstance, cat, dog, and horse), members of the family Muridae(including, for instance, rat or mouse), Guinea pig, or human. In oneembodiment, the animal may be an animal at risk of exposure to aninfectious disease, such as a disease caused by or associated with aviral, prokaryotic, or eukaryotic pathogen. In one embodiment, theanimal may be an animal in need of immunization against an antigen thatis associated with non-infectious disease, such as cancer. For instance,the antigen may be one that helps an animal mount an immune responsethat targets and eliminates cancer cells. The immune response may be ahumoral response (e.g., the immune response includes production ofantibody in response to an antigen), a cellular response (e.g. theactivation of phagocytes, antigen-specific cytotoxic T-lymphocytes, andthe release of cytokines in response to an antigen), or a combinationthereof.

In another embodiment, a method includes treating one or more symptomsof certain conditions in an animal. In one embodiment, a condition iscaused by infection by a virus or a microbe. As used herein, the term“infection” refers to the presence of and multiplication of a virus ormicrobe in the body of a subject. The infection can be clinicallyinapparent, or result in symptoms associated with disease caused by thevirus or microbe. The infection can be at an early stage, or at a latestage. In another embodiment, a condition is caused by a disease, suchas cancer. As used herein, the term “disease” refers to any deviationfrom or interruption of the normal structure or function of a part,organ, or system, or combination thereof, of a subject that ismanifested by a characteristic symptom or set of symptoms. The methodincludes administering an effective amount of a composition describedherein to an animal having or at risk of having a condition, or symptomsof a condition, and determining whether at least one symptom of thecondition is changed, preferably, reduced.

Treatment of symptoms associated with a condition can be prophylacticor, alternatively, can be initiated after the development of acondition. As used herein, the term “symptom” refers to objectiveevidence in a subject of a condition caused by infection by disease.Symptoms associated with conditions referred to herein and theevaluations of such symptoms are routine and known in the art. Treatmentthat is prophylactic, for instance, initiated before a subject manifestssymptoms of a condition, is referred to herein as treatment of a subjectthat is “at risk” of developing the condition. Accordingly,administration of a composition can be performed before, during, orafter the occurrence of the conditions described herein. Treatmentinitiated after the development of a condition may result in decreasingthe severity of the symptoms of one of the conditions, or completelyremoving the symptoms. In this aspect of the invention, an “effectiveamount” is an amount effective to prevent the manifestation of symptomsof a condition, decrease the severity of the symptoms of a condition,and/or completely remove the symptoms.

Also provided herein is a kit for immunizing an animal. The kit includesviral particles as described herein, where the second and/or thirdgenomic segments each independently include a coding region that encodesan antigen, in a suitable packaging material in an amount sufficient forat least one immunization. In one embodiment, the kit may include morethan one type of viral particle, e.g., the kit may include one viralparticle that encodes one or two antigens and a second viral particlethat encodes one or two other antigens. Optionally, other reagents suchas buffers and solutions needed to practice the invention are alsoincluded. Instructions for use of the packaged viral particles are alsotypically included.

As used herein, the phrase “packaging material” refers to one or morephysical structures used to house the contents of the kit. The packagingmaterial is constructed by known methods, preferably to provide asterile, contaminant-free environment. The packaging material has alabel which indicates that the viral particles can be used forimmunizing an animal. In addition, the packaging material containsinstructions indicating how the materials within the kit are employed toimmunize an animal. As used herein, the term “package” refers to a solidmatrix or material such as glass, plastic, paper, foil, and the like,capable of holding within fixed limits viral particles. Thus, forexample, a package can be a glass vial used to contain an appropriateamount of viral particles. “Instructions for use” typically include atangible expression describing the amount of viral particles, route ofadministration, and the like.

The term “and/or” means one or all of the listed elements or acombination of any two or more of the listed elements.

The words “preferred” and “preferably” refer to embodiments of theinvention that may afford certain benefits, under certain circumstances.However, other embodiments may also be preferred, under the same orother circumstances. Furthermore, the recitation of one or morepreferred embodiments does not imply that other embodiments are notuseful, and is not intended to exclude other embodiments from the scopeof the invention.

The terms “comprises” and variations thereof do not have a limitingmeaning where these terms appear in the description and claims.

Unless otherwise specified, “a,” “an,” “the,” and “at least one” areused interchangeably and mean one or more than one.

Also herein, the recitations of numerical ranges by endpoints includeall numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2,2.75, 3, 3.80, 4, 5, etc.).

For any method disclosed herein that includes discrete steps, the stepsmay be conducted in any feasible order. And, as appropriate, anycombination of two or more steps may be conducted simultaneously.

The above summary of the present invention is not intended to describeeach disclosed embodiment or every implementation of the presentinvention. The description that follows more particularly exemplifiesillustrative embodiments. In several places throughout the application,guidance is provided through lists of examples, which examples can beused in various combinations. In each instance, the recited list servesonly as a representative group and should not be interpreted as anexclusive list.

The present invention is illustrated by the following examples. It is tobe understood that the particular examples, materials, amounts, andprocedures are to be interpreted broadly in accordance with the scopeand spirit of the invention as set forth herein.

EXAMPLE 1

An Arenavirus-Based Vaccine Vector Carrying Influenza Virus AntigensConfers Long-Term Protection of Mice Against Lethal Influenza VirusChallenge

Pichinde virus (PICV) is a non-pathogenic arenavirus that has been usedas a model virus to study viral hemorrhagic fever infection. A reversegenetics system was previously developed to generate infectious PICVviruses from two plasmids that encode the viral large (L) and small (S)RNA segments (Lan et al., 2009, J Virol 83:6357-6362). In the currentstudy, a second-generation reverse genetics system was created toproduce recombinant infectious PICV viruses from 3 separate plasmidsthat are referred to as the tri-segmented PICV system. These recombinantviruses carry 3 viral genomic RNA segments that encode for all of theviral gene products as well as two foreign genes. This tri-segmentedPICV system can be used as a novel vaccine vector to deliver thehemagglutination (HA) and the nucleoprotein (NP) of the influenza virusA/PR8 strain. Mice immunized with these recombinant viruses areprotected against lethal influenza virus challenge as evidenced by thesurvival of the animals afforded by the high levels of HA neutralizingantibodies and NP-specific cytotoxic T lymphocyte (CTL) responses toviral infection. These tri-segmented recombinant PICV viruses do notinduce strong anti-PICV vector immunity, thus making them idealcandidates in a prime-boost vaccination strategy in order to inducecross-reactive immunity. In summary, a novel live vaccine vector hasbeen developed that can express multiple foreign antigens to inducestrong humoral and cell-mediated immunity and little anti-vectorimmunity in vivo.

Introduction

We have developed the only available reverse-genetics system to generateinfectious PICV viruses from plasmid transfection into appropriatemammalian cells (Lan et al., 2009, J Virol 83:6357-6362). This systemconsists of 2 plasmids to generate full-length genomic L and S RNAs froman engineered bacteriophage T7 regulatory (promoter/terminator) signalsand the hepatitis delta ribozyme sequence located upstream anddownstream of the PICV genomic sequences, respectively. When transfectedinto baby hamster's kidney epithelial cells that constitutively expressthe bacteriophage T7 RNA polymerase (BSRT7-5, a.k.a. BHK-T7), the L andS RNA segments of PICV are generated, from which all PICV viral geneproducts, including the L polymerase and NP nucleoprotein aretranscribed and translated in order to amplify the viral genome andgenerate infectious progeny virus particles. Infectious PICV viruses arereleased into supernatants and are isolated and amplified further byculturing on African green-monkey epithelial (Vero) cells.

As described herein a second-generation reverse genetics system has beendeveloped to produce recombinant infectious PICV viruses from 3 separateplasmids that are referred to as the tri-segmented PICV system. Theserecombinant viruses carry 3 viral genomic RNA segments that encode forall of the viral gene products as well as two foreign genes. Thistri-segmented PICV system can be used as a novel vaccine vector todeliver other viral antigens, such as those of the influenza virus.Briefly summarized, tri-segmented PICV viruses expressing thehemagglutination (HA) or the nucleoprotein (NP) of the influenza virusA/PR8 strain have been produced, and these tri-segmented PICV vaccinevectors can induce strong humoral and cell-mediated immunity, such as arobust production of influenza-specific neutralizing antibodies and astrong influenza-specific cytotoxic T lymphocyte (CTL) response invaccinated mice with little anti-PICV vector immunity, which makes themideal candidates for the prime-boost vaccination strategy in order toinduce long-lasting and cross-reactive immunity. Therefore, this novelPICV-based vaccine vector system described herein we have developedsatisfies all required criteria of an ideal viral vector, such assafety, induction of strong and durable cellular and humoral immuneresponses, no pre-existing immunity and lack of anti-vector immunity.

Materials and Methods

Construction of Plasmids Encoding the Engineered Pichinde Virus P18 SRNA Segments with Multiple-Cloning-Sites (MCS) Replacing Either GPC orNP Gene.

A reverse genetics system for PICV was previously developed bytransfection of 2 plasmids, encoding the L and S RNA segments inanti-genomic (ag) sense, into BHK-T7 cells (FIG. 1) (Lan et al., 2009, JVirol 83:6357-6362). The overlaping polymerase chain reaction (PCR)method was used to replace the open-reading-frame (ORF) of either theviral glycoprotein (GPC) or nucleoprotein (NP) gene with multiplecloning sites (MCS) in the S agRNA encoding plasmid. The resultingplasmids (FIG. 2A), P18S-GPC/MCS and P18S-MCS/NP, contain MCS with therestriction enzyme sequences (Nhe I-Mfe I-Acc65I-Kpn I- EcoR V-Xho I-SphI) that are introduced into these plasmids for the convenience ofcloning foreign genes (e.g., reporter genes and/or viral antigens).

-   Subcloning of GFP reporter gene, influenza HA or NP gene into    P18S-GPC/MCS vector. PCR was used to subclone the green fluorescence    protein (GFP) reporter gene and the influenza HA or NP gene from the    A/PR8 (H1N1) strain into the vector P18S-GFP/MCS between Nhe I and    Xho I sites, respectively. The amino acid sequence of the A/PR8    hemagglutinin can be found at Genbank Accession number NC_002017.1,    and the amino acid sequence of the A/PR8 nucleoprotein can be found    at Genbank Accession number NC_002019.1. The recombinant plasmid    constructs were confirmed by DNA sequencing. The resulting plasmids    (FIG. 5) are called P18S-GPC/GFP, P18S-GPC/H1, and P18S-GPC/NP,    respectively.

Recovery of Recombinant Tri-Segmented Pichinde Viruses Expressing GFP,Influenza HA or NP.

Recombinant viruses were recovered from plasmids by transfecting BSRT7-5(a.k.a. BHK-T7) cells with 3 plasmids expressing the full-length P18 LagRNA segment, a P18S segment with MCS in place of GPC (P18S-MCS/NP),and a P18S segment with GFP, HA, or NP gene in place of NP(P18S-GPC/GFP, P18S-GPC/N1, or P18S-GPC/NP) (FIG. 2B and FIG. 5). Theprocedures to generate recombinant PICV are essentially the same aspreviously described (FIG. 1B) (Lan et al., 2009, J Virol 83:6357-6362).Briefly, BHK-T7 cells were grown to 80% confluency and 4 hours beforetransfection the cells were washed and incubated with antibiotic-freemedia. For transfection, 2 ug of each plasmid was diluted in 250 ul ofOpti-MEM and incubated at room temperature for 15 minutes. An equalvolume of Opti-MEM with 10 ul of lipofectamine (Invitrogen, Lifetechnologies) was added and the mixture was incubated for 20 mins atroom temperature. Following incubation, the cells were transfected withthe plasmids and media was again replaced after 4 hours to removelipofectamine. After 48 hours of transfection, cell supernatants werecollected for plaque assay. Virus grown from individual plaques was usedto prepare stocks that were grown on BHK-21 cells and was stored at −80C.

Detection of Influenza Antigen Expression in Cells Infected withRecombinant Tri-Segmented PICV Vectors.

BHK-21 cells grown on coverslips were infected with wild type Pichindevirus (PICV) or recombinant PICV viruses expressing either the influenzaHA (rPICV-HA) or NP gene (rPICV-NP). After 24 hours of virus infection,cells were fixed with 4% paraformaldehyde for 15 mins at roomtemperature followed by 3 washes with phosphate buffer saline (PBS).Cells were then incubated in 0.1% Triton X-100 for 12 minutes followedby 1 hour incubation with primary antibody (mouse anti-NP influenza A).Cells were then washed and incubated with secondary antibody (anti mousealexa flour-647) for 1 hour at room temperature.

Mouse Immunization and Challenge.

Six- to eight-week old female C57BL/6 mice were obtained from CharlesRiver Laboratories and housed for at least 1 week for acclimatization.Mice were housed in microisolator cages in BSL-2 equipped animalfacility. Mice were injected with 100,000 pfu of rPICV virus in 100 ulof total volume through intra-peritoneal route. After 14 days of lastbooster, mice were challenged intra-nasally with 10 LD₅₀ (10,000 pfu) ofthe mouse-adapted influenza A/PR8 strain.

Analysis of Virus Specific CTL by Tetramer Staining.

Single-cell splenocytes were obtained and red blood cells (RBCs) werelysed using ACK lysis buffer. The splenocytes were then washed with FACSbuffer (PBS+2% FBS) twice. Cells (1×10⁵) were stained withCD8-PerCP-Cy5.5, CD3-APC and H-2Db-PE tetramer with the NP₃₆₆₋₃₇₄epitope ASNENME™ (SEQ ID NO:7) for 1 hr at room temperature. Afterincubation, cells were washed with FACS buffer three times and thelabeled cells were analyzed by flow cytometry. The FACS data wasanalyzed using FloJo software.

Hemagglutination Inhibition Assay.

Blood was collected after 14 days of each immunization and serum washarvested. Non-specific inhibitors were removed from the serum by anovernight treatment with 5 volumes of receptor destroying enzyme(Sigma), followed by 45 min incubation at 56° C. to inactivate theenzyme and serum. Each serum sample was then serially diluted in 25 ulof PBS and then mixed with an equal volume of PBS containing 4 HA unitsof A/PR8. After 15 min incubation at room temperature, 50 ul of 0.5%turkey RBC was added and the mixture was incubated for 1 hour at 4 Cbefore evaluation of agglutination. The titre was recorded as theinverse of the last dilution that inhibited agglutination.

Determination of Virus Titers in the Lungs.

To evaluate the replication of influenza A/PR8 in mice, lungs werecollected on day 6 of post challenge, weighed and homogenized in L15media. Tissue homogenate was centrifuged at 10,000 rpm for 10 mins andvirus was titrated in 12-well plate on monolayers of Madin-Darby caninekidney (MDCK) cells.

Results 1. Generation of Recombinant Tri-Segmented Pichinde VirusExpressing the GFP Reporter Gene.

A tri-segmented arenavirus system was first reported for LCMV by de laTorre group (Emonet et al., 2009,Proc Natl Acad Sci USA 106:3473-3478),which has generated infectious recombinant LCMV that packages 3 RNAsegments, one full-length L segment, and two S segments with deletion ineither GPC or NP. We have previously developed a reverse genetics systemfor the Pichinde virus (PICV) (Lan et al., 2009, J Virol 83:6357-6362),which is a prototypic arenavirus that is non-pathogenic in humans. Thissystem consists of 2 plasmids expressing the L and S segments of PICVwhen they are transfected into the BSRT7-5 (a.k.a. BHKT7) cells (FIG.1).

In the current study, a second-generation of the PICV reverse geneticsystem has been generated that consists of 3 plasmids that produce twodistinct S RNA segments, one carrying a deletion in GPC and the other Ssegment contains a deletion of the NP gene (FIG. 2). In place of thedeleted viral genes, we have inserted the multiple cloning sites (MCSs)(FIG. 2A). We then subcloned the GFP reporter gene into the P18 S2segment at the deleted GPC position using Nhe I/Kpn I sites within MCS,and generated infectious viruses from the supernatants of BSRT7-5 cellstransfected with three plasmids, one encoding the viral Z and L genes onthe full-length P18 L segment, and the other two S1 and S2 plasmids(i.e., P18 S1-MCS and P18 S2-GFP) expressing either the viral GPC genealone or both NP and GFP reporter genes (FIG. 2B). Stock viruses wereprepared from plaque-purified viruses. All plaques appeared green underfluorescence microscopy, demonstrating the successful recovery of therP18tri-GFP virus. In addition to Vero cells (data not shown),rP18tri-GFP virus infection expressed GFP in other permissive cells suchas BHK-21 and A549 (FIG. 3), and released more infectious progenyviruses that expressed GFP upon infection of a fresh culture of BHK-21target cells (FIG. 3), demonstrating the stability and integrity of therP18tri-GFP virus in cell cultures. Time course studies demonstratedthat GFP was strongly expressed after 12 h post-infection and that thecomplete life cycle of rP18tri-GFP was about 16 h as the concentrationof the rP18tri-GFP virus in the supernatants sharply increased at ˜16hpi (FIG. 4). In a preliminary experiment it was found that thePICV-tri-GFP virus vector can infect chicken embryonic fibroblasts (CEFcell line) in cell culture (data not shown).

Virus growths of the bi- and tri-segmented PICVs was compared byperforming the growth curve analysis of the rP2, rP18, and rP18tri-GFPon BHK-21 cells at the multiplicity of infection (moi) of 0.01. Asexpected, the known virulent rP18 virus grew slightly faster and betterthan rP2 by ˜0.5 log in virus titer. The tri-segmented rP18tri-GFP virusgrew at a similar kinetic as the rP2 and rP18 viruses albeit at ˜1 to1.5 log lowered virus titers (FIG. 4). In summary, the tri-segmentedPICV virus appears to be retarded in growth rate and titers as comparedto the bi-segmented rP2 and rP18 PICV viruses.

In order to determine the stability of the rP18tri-GFP virus, that is totest whether in vitro culturing of the rP18tri-GFP virus will select fora bi-segmented wild-type virus revertant that have lost GFP gene due togenome recombination, the rP18tri-GFP was passaged in the BHK-21 cellcultures continuously, and conducted plaque assay at various passages toexamine GFP expression in each of the plaques. At any tested passages(up to 23 passages), all plaques still expressed strong GFP reportergene levels, suggesting that the GFP reporter gene could be stablymaintained in the infectious viral particles even after extensivepassages in cell culture.

2. Generation of Recombinant Tri-Segmented Pichinde Virus ExpressingInfluenza Virus Antigens.

HA and NP genes of the influenza virus A/PR8/H1N1 strain weremolecularly cloned into the MSC of the P18 S2 plasmid, respectively(FIG. 5). The resulting P18 S2-HA and P18 S2-NP were separatelytransfected into BHK-T7 cells along with the full-length P18L andP18S1-GFP in order to generate the rP18tri-GFP/HA and rP18tri-GFP/NPviruses, respectively.

All infected cells (i.e., plaques) were green under fluorescencemicroscopy as the recombinant viruses carried the GFP reporter gene onthe P18S2-GFP segment (FIG. 6). In addition, HA and NP proteinexpressions were detected by immunofluorescence assay using specificanti-HA and anti-NP antibodies in Vero cells at 24 hr post infection(hpi) (FIG. 6). Taken together, we have shown that the tri-segmentedviruses can be made to express different foreign genes, including theGFP reporter gene and two different influenza viral antigens (HA and NP)upon cellular infection.

3. The rP18Tri-Based Influenza Vaccines Induce Protective Immunity inMice.

Previous work from our laboratory demonstrated that infection of micewith PICV even at high doses via different routes does not causediseases as PICV is cleared by 4 days post infection (dpi) (data notshown). Here, we have examined whether rP18tri-based vaccine vectors caninduce protective immunity in mice. C57BL6 mice were mock infected orinfected intra-nasally (i.n.) with a low dose (50 pfu) of A/PR8 or viai.p. route with 1×10⁵ pfu of rP18tri-GFP, rP18tri-GFP/HA, andrP18tri-GFP/NP viruses, respectively. Mice were boosted twice with thesame virus at the same dosage at day 14 and 28. Blood samples werecollected at different time points post priming and post boosting forneutralizing antibody titer determination. Mice were challenged at 14 dafter the 2^(nd) boost with a known lethal dose of A/PR8 (10×MLD50) andmonitored for body weight and disease symptoms for up to 21 days. Allchallenged animals that have previously received PBS or rP18tri-GFPvaccination reached terminal points by 7 dpi, whereas all micevaccinated with rP18tri-GFP/HA were completely protected from lethalinfluenza virus infection. Two out of three mice vaccinated withrP18tri-GFP/NP survived (FIG. 7). For rP18tri-GFP/HA vaccination,challenged mice maintained body weight, while the two survivors in therP18tri-GFP/NP group showed a slight body weight loss at the early stagebut quickly recovered (data now shown). Consistent with the mortalitydata, mice vaccinated with HA or NP had minimal pathological changes inthe lungs at 3 dpi, compared to those with PBS or vector alone.Similarly, virus titers in the lungs at 3 dpi also correlated withprotection. A/PR8 virus replicated to high levels in mouse lungs at 3dpi when the animals were vaccinated with mock or the rP18tri-GFP vectoralone. In contrast, virus titer was not detectable in the rP18tri-GFP/HAvaccination group and was significantly reduced in the rP18tri-GFP/NPgroup.

Taken together, these results suggest that the rP18tri-based vaccinevector can induce protective immunity from lethal influenza virusinfection in mice. Consistent with previous influenza vaccine studies,HA vaccination can induce a complete protection from influenzavirus-induced disease while NP vaccination normally leads to partialprotection by reducing disease severity and controlling virusreplication in vivo.

4. The rP18Tri-GFP/HA Vaccine Induces High Neutralizing Antibody Titersin Mice.

The hemagglutination inhibition (HAI) titers was measured in peripheralblood collected from vaccinated mice at different time points: 14 dpost-prime, 7 d and 14 d post-1^(st) boost, and 14 d post-2^(nd)-boost(FIG. 8). As expected, HAI titers from the rP18tri-GFP andrP18tri-GFP/NP groups did not surpass the background level, becauseneither GFP nor NP is expected to induce HA-specific neutralizingantibodies. In contrast, HAI titers from the rP18tri-GFP/HA vaccinationgroup reached the level of 40 at 14 d post-prime, and increasedsubstantially after boosting (FIG. 8), suggesting that therP18tri-GFP/HA vaccine can induce high levels of neutralizing antibodytiters in vivo. As a threshold HAI titer of >=40 is generally used as ameasure of success, i.e., in providing 50% reduction in the risk ofinfluenza (seroprotective titer) (Reber et al., 2013, Expert RevVaccines 12:519-536), the HAI data suggests that a single dose ofrP18tri-GFP/HA may be sufficient to confer protection.

HAI titers generated by different vaccination routes were compared (FIG.9). Mice were vaccinated with rP18tri-GFP/HA by one prime and one boostvia intraperitoneal (i.p.), intramuscular (i.m.), or intranasal (i.n.)route. All three routes of immunization elicited significant levels ofhumoral response as demonstrated by relatively high HAI titers overall.However, it appears that the HAI titres in sera of the i.m and i.pinjected routes were relatively higher than those of the i.n. group.Regardless, these HAI titers were high enough to confer completeprotection against a lethal challenge with the PR8 influenza virus. Asshown in FIG. 10 there was no significant weight loss observed in thevaccinated animals.

5. The rP18Tri-GFP/NP Vaccine Induces Strong CTL Responses in Mice.

NP is an intracellular viral protein and is known to induce CD8+ T cellresponse against it. Whether the rP18tri-GFP/NP vaccine can induce CTLresponses was examined by conducting NP-specific tetramer analysis ofsplenic cells and PBMCs at different time points after prime and boostwith rP18tri-GFP/NP intraperitoneally. As shown in FIG. 11 (top),NP-specific CD8+ and CD44+ T cells were detected in the rP18tri-GFP/NPvaccinated group even at 7 d post-prime, at comparable (if not higher)level to a low dose of PR8 infection, which increased further at 7 dpost-boost, and were still present at 14 d post-boost (FIG. 11 middle).We also explored the effect of different inoculation routes on CTLresponses. As shown in FIG. 12 both i.m. and i.p. inoculation routesinduced stronger CTL responses than the i.n. route of infection.Collectively, our data demonstrate that rP18tri-based vector can inducestrong CTL responses in vivo and that the intramuscular andintraperitoneal routes are better than intranasal route in inducingstrong CTL responses. Since the i.m route elicited optimal humoral and Tcell responses, subsequent experiments were conducted following the i.mroute of immunization.

6. Can rP18Tri-Based Vaccine Trigger Long-Lasting Immunity?

The potential of the rP18tri-GFP/HA vaccine vector to induce longlasting immunity was evaluated. C57BL6 mice were immunized twice at aninterval of 4 wks and were challenged either 4 or 8 wks after secondimmunization with a lethal dose of the A/PR/8 (H1N1) virus. Serumsamples analysed for HAI titres at days 14 and 30 post prime and days 30and 60 post boost demonstrated a strong neutralizing antibody titer(FIG. 13A). The challenged virus A/PR/8 replicated well in the lungs ofmock-vaccinated mice whereas there was no detectable virus titer in thelungs of vaccinated mice that were challenged after 4 wks ofvaccination. However, there were significantly less viral titers in thelungs of immunized mice that were challenged after 8 wks of vaccination(FIG. 13B). As shown in FIG. 13C, complete protection was observed asthere was no significant weight loss in the rP18tri-GFP/HA immunizedmice challenged after either 4 wks or 8 wks of vaccination.Consistently, there were no significant pathological changes in thelungs of immunized mice (FIG. 13D). Overall, the levels of immunity andprotection were comparable with those seen at 2 wks after immunization.

Single Immunization with rP18tri-GFP/HA Induced Protection AgainstLethal H1N1 Challenge

C57BL6 mice were immunized with rP18triGFP-HA through intramuscularroute. Remarkably, a single dose of rP18tri-GFP/HA induced completeprotection against lethal infection with the mouse-adapted A/PR/8 (H1N1)virus. As shown in FIG. 14B, rP18tri-GFP vaccinated control micecontinued to lose weight after challenge until they required euthanasia.The results also demonstrated that a single immunization with a minimumdose of 10³ pfu of the rP18tri-GFP/HA vector elicited a robusthemagglutination inhibiting (HI) Ab response and was sufficient toconfer protection (FIG. 14A). Histological analyses of the lung sectionconducted 5-6 days after challenge revealed a significant reduction ininflammation in mice vaccinated with rP18tri-GFP/HA whereas micevaccinated with rP18tri-GFP showed massive infiltration of inflammatorycells into the lung, lung congestion and intra-alveolar edema (FIG.14C).

Generation and Characterization of rP18Tri-Based Virus Expressing DualInfluenza Viral Protein Antigens

To obtain the rP18tri-based vector expressing influenza HA and NP or HA1and HA3, DNA encoding the respective influenza virus genes was insertedinto plasmid vectors P18S-GPC/MCS and P18S-MCS/NP accordingly. TheRT-PCR amplified HAL NP and HA3 were cloned and each clone was sequenceverified. The resulting plasmids were designated as P18S-GPC/HA1,P18S-NP1/NP and P18S-HA3/NP. The recombinant virus expressing dualantigens (influenza HA1 and NP1) was then rescued by co-transfecting 3plasmids expressing the full-length P18 L agRNA segment, P18S-GPC/HA1and P18S-NP1/NP. Simultaneously, a similar 3-segmented recombinant viruswith swapped influenza genes was also generated by co-transfecting thefull-length P18 L agRNA segment, P18S-GPC/NP1 and P18S-HAl/NP.Similarly, recombinant virus expressing dual antigens (influenza HA1 andHA3) was rescued by co-transfecting 3 plasmids expressing thefull-length P18 L agRNA segment, P18S-GPC/HAland P18S-HA3/NP.

Interestingly, rP18tri-HANP had lower titres than rP18tri-NP/HA andformed smaller plaques in Vero cells (FIG. 15A). However, the expressionlevel of the influenza viral protein antigens was higher in therP18tri-HA/NP as compared to the rP18tri-NP/HA (FIG. 15B).

rP18Tri-Based Vector Encoding Multiple Influenza Viral Protein Antigens(HA and NP) Induced Both Humoral and T Cell Responses in Immunized Mice.

rP18tri-based vector encoding both influenza viral HA and NPsuccessfully induced potent humoral and T cell responses in mice thatwere immunized with 10⁴ pfu/ml of either the rP18tri-HA/NP orrP18tri-NP/HA vector. Peripheral blood and spleen of mice were used tomonitor the levels of T cell responses to immunodominant NP336 epitopeusing an MEW class I tetramer. Anti-NP-specific CD8+ T cells weredetected in peripheral blood and splenocytes of immunized mice. On day 7post-immunization, mice had a significantly greater percentage ofNP-specific T cells (FIG. 16A). Tetramer positive CD8+ T cells peakedafter 7 days of boost and were maintained at a resting level until 14days post boost (FIG. 16B). Both of the vaccine candidates also eliciteda strong humoral response that was assessed by hemagglutinationinhibition titre in sera collected at 14 days post prime and 14 dayspost boost. The HI titres were increased post boost in mice immunizedwith rP18tri-HA/NP whereas no such elevation was observed in miceimmunized with the rP18tri-NP/HA (FIG. 16C).

Taken altogether, the rtriP18-HA/NP vaccine elicited a stronger immuneresponse in comparison to the rP18tri-NP/HA vaccine. The better levelsof immune response of the rP18tri-HA/NP could be attributed to a higherexpression level of the influenza viral antigens (FIG. 15B).Nevertheless, both the vector vaccines rP18tri-HA/NP and rP18tri-NP/HAconferred complete protection against a lethal challenge of the A/PR/8influenza virus. No disease symptoms were observed in the immunizedmice. As shown in FIG. 17A, no significant histological changes could bedetected in the lungs of mice immunized with either of the vaccinecandidates. In contrast, infiltration of inflammatory cells was observedin lungs of mice vaccinated with the rP18tri-GFP. Viral titres in lungswere analyzed to determine the replication of the challenged virus atday 3 and 6 post infection. The mock-vaccinated mice showed high viraltitres while mice vaccinated with the rP18tri-HA/NP and rP18tri-NP/HAshowed no detectable titres at day 6 and one out of three mice in eachgroup showed titres of 3×10³ pfu/ml and 5×10³ pfu/ml respectively at day3 post infection (FIG. 17B). Mice vaccinated with either of the vaccinecandidate showed no weight loss (FIG. 17C).

rP18Tri-Based Vector Encoding Multiple Influenza Antigens (HA1 and HA3)Induced Protection Against Dual Challenges

Different groups of C57BL6 female mice were immunized with two dosagesof either the rtriP18-based vector vaccine encoding only HA of theH1-subtyped virus (rP18tri-HA1) or HA of the H3-subtyped (rP18tri-HA3)or both HA of the H1 subtype and the H3 subtype (rP18tri-HA1/HA3). Theresults indicated that the first dose of the rtriP18-based vectorencoding HA1 and HA3 induced antibody responses to both the encodedinfluenza viral antigens as revealed by robust HI titres for PR8 andX31. The second dose of the vaccine further boosted the antibodyresponse. Interestingly, the HI titres induced for PR8 (H1) and X31 (H3)by rP18tri encoding both HA1 and HA3 were comparable to that induced bythe rtriP18-based vector encoding only HA1 or HA3 (FIG. 18A). Similarresults were obtained in Balb/c mice (FIG. 18B). Upon challenge with alethal dose of the PR8 (H1N1) or X31 (H3N2) virus, mice immunized withthe rP18tri-based vector encoding both HA1 and HA3 (rP18tri-HA1/HA3)were protected against both viral challenges. The protective efficacy ofthe rP18tri-HA1/HA3 in preventing the replication of the challengedviruses was evaluated on day 5 following virus challenge. The challengedviruses PR8 (H1N1) and X31 (H3N2) replicated well in the lungs ofmock-vaccinated mice with a mean titres of 10⁵ and 10⁴pfu/m1respectively. Immunization with the rP18tri-HA1/HA3 protected mice fromboth of the viral challenges as there was no loss of body weight andnone of the immunized mice had any detectable viral titres in the lungs(FIG. 19A and B).

rP18Tri-Based Flu Vaccines do not Induce Anti-Vector Immunity.

A perceived weakness of live viral vector is the anti-vector immunity,which impairs the immune responses induced by the same vector andexcludes its usage in a prime-boost vaccination strategy. PICV haslittle to no pre-existing immunity in general population (Trapido etal., 1971, Am J Trop Med Hyg 20:631-641). We determined the effect ofanti-vector immunity against PICV vector by measuring the levels ofantibody and CTL responses by prime-boost with the same vector.Surprisingly, we found no evidence for anti-vector immunity againstrPICVtri vector. Shown in FIG. 8, boosting with the same vector furthersubstantially increased the HAI titers, suggesting that HA-specifichumoral responses increased after the 2^(nd) and even 3^(rd) boosting.Similar finding was observed for the CTL responses. The NP-specific CD8and CD44 T cells in both spleen and PBMCs increased after boosting (FIG.20). Taken together, our data suggest that rPICV vector does not inducestrong anti-vector immunity and thus can be repeatedly used in theprime-boost vaccine strategy.

Summary

A novel PICV reverse genetic system has been developed to generatetri-segmented recombinant PICV viruses that can encode up to two foreigngenes of interest. These rP18tri-based recombinant viruses can expressthe influenza HA and/or NP genes along with the GFP reporter gene intarget cells. When tested in mice, the rP18tri-GFP/HA virus can inducehigh levels of HA-specific neutralizing antibodies, while therP18tri-GFP/NP virus can induce strong NP-specific CTL responses. Inaddition, the rP18tri-based vector expressing dual influenza viralantigens HA and NP successfully induced both humoral and T cellresponses that conferred complete protection in immunized mice against alethal challenge with the A/PR8. More importantly, the triP18- basedvector expressing HA of two different influenza virus subtypes i.e PR8(H1N1) and X31 (H3N2) conferred protection against both of the viralchallenges, demonstrating the prowess and versatility of these novelvaccine vectors against pathogenic influenza viruses. It is alsoimportant to note that there is no pre-existing immunity against PICVvector and that animals immunized with the tri-segmented PICV virusesgenerate little anti-PICV vector immunity, making this an ideal vectorvaccine platform for inducing potent, long-lasting and cross-reactiveimmunity upon repeated vaccination in the event that a prime-boostvaccination strategy is needed.

EXAMPLE 2

The Viological Role of NP Exoribonuclease in Arenavirus Infection InVitro and In Vivo

Arenavirus NP RNase activity is important for type I interferon (IFN)suppression but its biological role(s) have not been well characterized.Recombinant Pichinde viruses with RNase catalytic mutations induced highlevels of IFNs and grew poorly in IFN-competent cells, and, wheninfecting guinea pigs, stimulated strong IFN responses, failed toreplicate productively, and generated wild-type revertants. Thus, the NPRNase activity is essential for the IFN suppression and establishingproductive replication early in arenavirus infection.

Arenaviruses include several hemorrhagic fever (HF)-causing agents(e.g., Lassa virus-LASV), with limited preventative or therapeuticmeasures (McLay et al., 2013, Antiviral Res 97:81-92, McLay et al.,2014, J Gen Virol 95:1-15). Arenavirus pathogenesis is associated withhigh viremia and generalized immune suppression, the mechanism of whichis poorly understood. It has been shown that viral NP can effectivelymediate Type-I IFN suppression via its exoribonuclease (RNase) function(Jiang et al., 2013, J Biol Chem 288:16949-16959, Martinez-Sobrido etal., 2007, J Virol 81:12696-12703, Martinez-Sobrido et al., 2006, JVirol 80:9192-9199, Qi et al., 2010, Nature 468:779-783, Hastie et al.,2011, Proc Natl Acad Sci USA 108:2396-2401, Hastie et al., 2012, PLoSONE 7:e44211). However, the role of the NP RNase activity in mediatinghost immune suppression in vivo has not been well characterized. In thisstudy, Pichinde virus (PICV) infection of guinea pigs was used as asurrogate model of arenavirus hemorrhagic fevers (HFs) (Aronson et al.,1994, Am J Pathol 145:228-235, Lan et al., 2009, J Virol 83:6357-6362)to characterize the role of the NP RNase in viral infection and host IFNresponses. Single alanine substitution at each of the RNase catalyticresidues (D380A, E382A, D525A, H520A, and D457A) could abolish the NPability to suppress Sendai virus-induced IFNβ activation by a luciferase(LUC) reporter assay (FIG. 21A), corroborating our previous observationswith LASV NP (Qi et al., 2010, Nature 468:779-783). Using our developedrP18 PICV reverse genetics system (Lan et al., 2009, J Virol83:6357-6362), we successfully generated recombinant viruses carryingindividual RNase mutations, which were confirmed by sequencing. The 5RNase mutants, together with the WT rP18 that causes virulent infectionin guinea pigs, and PICV rP2 that causes avirulent infection, were usedto infect human airway epithelial A549 cells at MOI=1. Type-I IFNproductions at 12 hpi and 24 hpi were quantified by the rNDV-GFPbiological assay (Park et al., 2003, J Virol 77:1501-1511). Both rP2 andrP18 produced low levels of IFNs similar to mock infection, asdemonstrated by the high levels of GFP expression (FIG. 21B). This isnot surprising as NP proteins of both strains encode a functional RNasedomain and do not seem to differ in their ability to suppress IFNproduction (Lan et al., 2008, Arch Virol 153:1241-1250). In contrast,the RNase mutants produced significantly more IFNs, as shown by thegreatly reduced GFP expression (FIG. 21B). Thus, the NP RNase activityis required for the efficient inhibition of type I IFNs invirus-infected cells.

Viral growth kinetics was determined in the IFN-deficient Vero cells andIFN-competent A549 cells at moi=0.01. All 5 mutants replicated well inVero cells, reaching 10⁶ pfu/ml at 48 hpi, albeit at ˜0.5-1 log lowerthan the WT rP18 (FIG. 22A, left). In sharp contrast, these RNase mutantviruses barely grew in A549 cells (FIG. 22A, right). Our results suggestthat the NP RNase activity is non-essential for the basic virus lifecycle but is required for productive viral replication in theIFN-competent cells, consistent with the recently published data on theLASV double-mutant (D389A/G392A) (Carnec et al., 2011, J Virol85:12093-12097).

To compare viral virulence in vivo, we infected 6 Hartley outbred guineapigs intraperitoneally with 1×10⁴ pfu of each virus as previouslydescribed (Lan et al., 2009, J Virol 83:6357-6362, Kumar et al., 2012,Virology 433:97-103, McLay et al., 2013, J Virol 87:6635-6643). Allprocedures were approved by the Institutional Animal Care and UseCommittee (IACUC) at University of Minnesota. Animals were monitoreddaily for body weight and disease signs for up to 18 days, and wereeuthanized when they reached the predetermined terminal points(moribund), such as more than 30% weight loss compared to a nomogram orrectal temperature below 38° C. in combination with continuing weightloss. As expected, all rP2-infected animals survived and cleared thevirus, while rP18-infected animals developed early onset of fever,showed significantly decreased body weight starting at 7 dpi, andreached terminal points by 13 dpi (FIG. 22B). The RNase mutant virusescaused variable disease outcomes (FIG. 22B and Table 1). All E382Amutant virus-infected animals survived without any evidence of bodyweight loss, while animals infected with each of 3 other mutant viruses(D525A, H520A, and D457A) showed various degrees of attenuation (FIG.22B). Most D380A-infected animals, however, succumbed to the infectionalbeit at a later time point than the rP18. To determine whether WTrevertants occurred, we measured the viremia levels at end point andsequenced the viruses (Table 1). As expected, moribund animals wereassociated with high viremia while all those who survived had very lowto undetectable levels of virus infection. Without exception, virusesisolated from mutants-infected animals had reverted back to the WT NPsequence (Table 1). This is highly unlikely due to PCR contamination orsequencing error as the viruses isolated from other mutantvirus-infected animals conducted at the same time still contain theexpected mutations (data not shown). We therefore believe that thedisease phenotypes are caused by WT viruses, including the WTrevertants, and that the disease severity is determined by how fast thereversion occurs in vivo (a few surviving animals were found to carrylow levels of WT virus at day 18). Our results strongly suggest anessential role of the NP RNase activity in arenaviral infection in vivo.

TABLE 1 Guinea pigs infected with the respective recombinant PICVs.Recombinant Viremia¹ PICV strains Disease (PFU/ml) Viral sequences² rP2³survived ND⁴ rP18 moribund 2.00E+06 moribund 5.80E+05 moribund 2.80E+05moribund 1.80E+06 moribund 2.30E+05 moribund 8.00E+05 D380A moribund5.00E+04 WT reversion moribund 3.10E+05 WT reversion moribund 3.30E+06WT reversion moribund 1.60E+04 WT reversion moribund 5.00E+04 WTreversion survived ND E382A survived ND survived 2.13E+03 WT reversionsurvived ND survived 9.00E+02 WT reversion survived ND survived ND D525Amoribund 7.50E+04 WT reversion moribund 6.25E+04 WT reversion moribund1.75E+05 WT reversion moribund 1.40E+04 WT reversion moribund 2.40E+04WT reversion moribund 2.10E+05 WT reversion H520A moribund 5.75E+05 WTreversion survived ND moribund 7.50E+05 WT reversion moribund NDmoribund ND moribund ND D457A survived ND survived ND survived NDsurvived 6.20E+02 WT reversion moribund 5.50E+04 WT reversion moribund1.10E+05 WT reversion ¹Virus titers in the blood collected at the timeof euthanization when animals reached terminal points or at day 18 postinfection. ²Sequences of viruses isolated from animals at the time ofeuthanization when animals reached terminal points or at day 18 postinfection. ³All 6 animals in the rP2-infected group survived with nodetectable viremia at day 18 post infection. ⁴ND, not detectable byplaque assay

To examine the role(s) of RNase in early viral infection and host innateimmune responses in infected animals, we monitored viral spread andquantified innate antiviral genes at 1 dpi and 3 dpi. At day 1, viruseswere detected at low levels in the livers and spleens of rP18-infectedanimals and in the livers of some animals infected with rP2 and theRNase mutant viruses (FIG. 23A). At day 3, both rP18 and rP2 replicatedto relatively high levels in both livers and spleens. In contrast, RNasemutant viruses appeared to be cleared from the livers and only detectedin a few spleens at very low levels (FIG. 23A), Representative innateimmune response genes, IFN-α1, IFN-β1, ISG15, IRF7, RIG-I, and MDA5, inthe peritoneal cavity cells at day 1 were quantified by qRT-PCR (FIG.23B). These genes were highly activated by the RNase mutant viruses butnot by the WT viruses (rP2 and rP18), demonstrating that NP RNase isrequired for arenavirus-induced innate immune suppression in vivo.

In summary, our study with recombinant PICV RNase mutants has not onlyconfirmed the important role of NP RNase in type I IFNs suppression andviral replication in vitro, but also provided unequivocal evidence forits essential role in early innate immune suppression to allowestablishment of a productive infection in vivo. Given that the NPRNase-dependent IFN suppression mechanism is conserved amongarenaviruses (Jiang et al., 2013, J Biol Chem 288:16949-16959), ourresults can be extrapolated to other arenaviral pathogens and implicateNP RNase as an ideal target for antivirals development.

EXAMPLE 3 The Arenavirus-Based Vaccine Vector Delivers Two Antigens andConfers Immunity to each of the Antigens

1. Generation of rP18tri Live Vaccine Vectors Expressing Dual InfluenzaAntigens HA and NP

Example 1 discloses a replication-competent tri-segmented rPICV system(rP18tri) and using this rP18tri vector to express either the influenzaHA (abbreviated as H) or NP antigen (P), it was demonstrated that thevector can induce strong antibody and CTL responses in mice. Todetermine whether a single rP18tri virion particle can be used todeliver both HA and NP genes, S1 and S2 viral genomic RNA segments weregenerated to encode the influenza HA and NP genes, respectively. Byusing different combinations of plasmids in the transfection reactions,we have successfully generated live vaccine vectors, rP18tri-P/H andrP18tri-H/P, which encode HA and NP genes on different S segments asillustrated in FIG. 24A. As a control vector, the rP18tri vectorencoding the eGFP reporter gene on both the S1 and S2segments—rP18tri-G/G—was also generated. To examine the antigensexpression, Vero cells were infected with rP18tri-G/G, rP18tri-P/H, andrP18tri-H/P respectively. At 24 hpi, cells were examined for theexpression of HA and NP by IFA using the mouse anti-HA and anti-NPantibodies, respectively, followed by detection with a PE-conjugatedanti-mouse antibody. Expression of both HA and NP proteins was detectedin cells infected with rP18tri-P/H and rP18tri-H/P but not in thoseinfected with the vector control rP18tri-G/G. The lower number of cellsexpressing HA and NP by the rP18tri-H/P infection than by therP18tri-P/H is due to a lower viral titer used in the infection (FIG.24B). Subsequent viral growth analysis in BHK-21 cells at moi of 0.01suggests that rP18tri-P/H and rP18tri-H/P replicate at similar kineticsand are <0.5 log lower than the vector control rP18tri-G/G (FIG. 24C).

2. The HA/NP Dual Antigen Vaccine Vectors can Induce Protective Immunityin Mice.

To test the protective immunity of rP18tri-P/H and rP18tri-H/P livevectors in vivo, we immunized a group of mice (n>=3) with therP18tri-G/G control vector, rP18tri-P/H, or rP18tri-H/P at 1×10⁴ pfuthrough the IM route, boosted with the same vectors 14 days later, andchallenged with a lethal dose of the mouse-adapted A/PR8 influenza virus(10×MLD₅₀) 14 days after vaccination. All the control vector-immunizedmice succumbed to the infection by day 6, while all mice receivingrP18tri-P/H or rP18tri-H/P survived the challenge without any diseasesigns or body weight loss (FIG. 25A). Compared to the controlvector-immunized mice that had high viral titers (2-10 ×10⁵ pfu/g) inthe lungs at 3 and 6 dpi, the rP18tri-P/H or rP18tri-H/P-immunized mice(n=3 in each group) had no detectable viruses at 6 dpi and only one ofthree had a relatively low level of viruses (<5×10³ pfu/g) at 3 dpi(FIG. 25B). Our data suggest that the dual antigen expressing viralvectors can induce strong protective immunity against influenza virus,which is conferred by HA-specific neutralizing antibody.

3. The H1/H3 Dual Antigen Vector Induces Balanced HA NeutralizingAntibodies.

Whether two different HA subtypes can be used to induce neutralizingantibodies at equal efficiency was determined. Toward this end, we havecloned the H3 HA gene of the A/x31 (H3N2 subtype of the influenza virus)to the S2 segment and when transfected with the full-length L segment,and the S1 segment encoding the H1 HA of A/PR8, live rP18tri-H3/H1vector can be generated (FIG. 26A). As controls, we have also generatedthe rP18tri vectors encoding eGFP on the S2 segment and either H1 or H3on the S1 segment, respectively called rP18tri-G/H1 or rP18tri-G/H3(FIG. 26A). To test their immunogenicity, groups of C57BL6 mice (n=3 pergroup) were primed and boosted with the three vectors respectively at a14-day interval through the IM route. Blood collected 14 days afterprime and boost were measured for the levels of neutralizing antibodyagainst A/PR8/H1N1 and A/x31/H3N2 (FIG. 26B top panel). The rP18trivectors encoding single HA subtypes, rP18tri-G/H1 and rP18tri-G/H3, eachinduced strong homosubtypic neutralizing antibodies that increase over aboost dose, but did not induce detectable levels of cross-reactiveantibodies after prime or boost. The rP18tri-H1/H3 dual antigen vectorinduced both H1 and H3 neutralizing antibodies that increased upon abooster dose and that the level of each neutralizing antibody wascomparable to that induced by the respective single antigen vectors(FIG. 26B top). Similar findings were obtained with Balb/c mice (FIG.26B bottom). Taken together, our data strongly suggest that the H1/H3dual antigen vector induces balanced neutralizing antibodies againstboth antigens in mice. In other words, there is no preference (skewing)of production of neutralizing antibodies against one or the other HAantigen.

4. Induction of Heterosubtypic Neutralizing Antibodies by aPrime-and-Boost Strategy with Different HA Subtypes

Recent studies have suggested that broadly neutralizing antibodies canbe generated by prime-and-boost vaccinations or sequential infections(Wei et al., 2010, Science 329:1060-1064, Wei et al., 2012, Sci TranslMed 4:147ra114, Miller et al., 2013, Sci Transl Med 5:198ra107, Wrammertet al., 2011, J Exp Med 208:181-193, Krammer et al., 2012, J Virol86:10302-10307, Miller et al., 2013, J Infect Dis 207:98-105, Margine etal., 2013, J Virol 87:4728-4737). As the rP18tri vector enhances immuneresponses upon a booster dose, whether it can induce cross-reactiveimmunity using a prime and boost strategy with different HA subtypes wastested. Toward this end, live rP18tri-P/H1 and rP18tri-P/H3 vectors weregenerated, each encoding A/PR8 NP, a conserved viral protein (seeGenBank Accession number NP_040982.1) that is known to elicit T cellresponses, together with either H1 (A/PR8) or H3 (×31) HA (FIG. 27A).Mice were primed with rP18tri-P/H1, boosted with rP18tri-P/H3, andboosted again with rP18tri-P/H1, each time at a 14-d interval. Asexpected, NP-specific CTLs were detected after prime (7 dpp),significantly increased upon a booster dose (7 dpb), and still remainedat high levels (3-5%) after a second booster dose (FIG. 27B). Blood werecollected 14 days after each administration and tested for the levels ofneutralizing antibodies against A/PR8, A/x31, and A/WSN. Neutralizingantibodies specific to homologous viruses A/PR8 (H1) and A/31 (H3) werehighly induced upon the exposure of the same HA antigens and were notgenerally enhanced by the heterologous HA boosting, whereas neutralizingantibodies against heterosubtypic virus A/WSN (H1) were steadilyincreased upon the booster doses (FIG. 27C). These immunized mice showeda significantly improved survival after a lethal challenge with theheterosubtypic virus A/WSN (FIG. 27D). Taken together, our data suggestthat prime-and-boost with heterologous HA subtypes using the rP18trivector can elicit cross-reactive neutralizing antibodies and cause crossprotection against heterosubtypic influenza virus challenge.

5. The rP18Tri Vector can Induce Both Humoral and T Cell ResponsesThrough Oral Route.

To determine whether the rP18tri vector can be conveniently giventhrough oral route, C57BL6 mice were immunized with 1×10⁴ pfu of therP18tri-G vector (n=3) or rP18tri-P/H (n=4) through oral gavage andboosted with the same vector 42 days later. The NP tetramer-positiveeffector T cells (CD8⁺CD44^(high)) at 7 days post prime and post boostwere measured by the established NP tetramer analysis. At 7 days postprime, two out of three tested mice from the rP18tri-P/H-immunized micehad NP⁺CD8⁺CD44^(high) cells (1.24% and 0.55%) that were clearly higherthan the background level seen in the vector-immunized mice (0.13% and0.09%). The NP-specific effector cells increased significantly at 7 dayspost boost, ranging from 5.7 to 7.1% in all 4 immunized mice (FIG. 28A).Similar patterns were observed for the neutralizing antibodies. Two outof four immunized mice showed a positive HAI titer (HAI=20) at 14 dayspost-prime. At 42 days post-prime, all 4 mice showed HAI titers inaverage of 40. After a boost dose, the HAI titer increased significantlyfor all 4 mice, ranging from 80 to 160 (FIG. 28B). Taken together, ourdata strongly suggest that the rP18tri vector can induce high levels ofboth humoral and T cell responses through oral route after a boosterdose.

6. Inactivated rP18Tri Vector can Induce both Humoral and T CellResponses.

Whether inactivated rP18tri vector can induce vaccine immunity wasdetermined. Live or hydrogen peroxide-treated rP18tri-P/H vaccine vector(i.e., inactivated vaccine vector) was used to immunize mice in 2 dosesat a 34-day interval. Neither neutralizing antibodies nor NP-specific Tcells were detected immediately after prime (7 days post-prime, 7 dpp).However, high levels of NP-specific effector T cells were detected aftera booster dose (7 days post-boost, 7 dpb) with the inactivated vaccinevector (FIG. 29, left). Neutralizing antibodies were detected at a lowlevel (HAI=20) in all three mice 34 days after prime (34 dpp), andincreased significantly after a booster dose (14 days post-boost, 14dpb), ranging from 40 to 80 with the inactivated vaccine vector (FIG.29, right). It is worth noting that the levels of both T cell andhumoral responses induced by the chemically inactivated rP18tri-P/Hvaccine vector are significantly lower than those induced by the livevaccine vector. Nevertheless, that the inactivated rP18tri-P/H can stillinduce both humoral and T cell responses after a booster dose suggeststhe versatility of this viral vector as a vaccine platform as both alive and inactivated vaccines.

7. The rP18Tri Vector Induces Protective Immunity Against Virulent P18Virus in a Guinea Pig Model.

WT P18 virus was obtained after serial passages of the Pichinde virus(PICV) in guinea pigs and causes a hemorrhagic fever-like disease in theanimals, which has been used as a safe surrogate model for studyingpathogenesis of Lassa fever virus infection in humans (Jahrling et al.,1981, Infect Immun 32:872-880, Aronson et al., 1994, Am J Pathol145:228-235, Liang et al., 2009, Ann NY Acad Sci 1171 Suppl 1:E65-74).Compared to WT P18 virus, the tri-segmented rP18tri vectors grow atleast 1 log lower in vitro. We also determined the virulence potentialof the rP18tri-G vector in guinea pigs. Guinea pigs infected with 1×10⁴pfu of rP18 virus developed early onset fever, rapidly lost weight after7 days post-infection, and reached terminal points by day 14 (FIG. 30A).In contrast, guinea pigs infected with 1×10⁶ pfu of rP18tri-G (100-foldhigher titer than rP18 control) showed a similar body weight growth asthe mock-infected animals and did not experience fever of more than 1day (FIG. 30A).

Whether this non-pathogenic rP18tri vectors could induce protectiveimmunity against a lethal rP18 challenge in guinea pigs was determined.Guinea pigs were injected with either phosphor buffer saline (PBS) torepresent mock infection or with 1×10⁴ pfu of rP18tri-G through IP routeand, 14 days later, challenged with a lethal dose of rP18 virus.PBS-immunized guinea pigs (n=3) soon developed fevers and lost bodyweight significantly and all animals reached predetermined terminalpoints by day 11 (FIG. 30B). In contrast, rP18tri-G-immunized guineapigs (n=3) were completely protected with no appreciable body weightloss (FIG. 30B). The protective immunity against PICV is likely mediatedby T cell responses as neutralizing antibodies have yet to develop atthe time of challenge. Previous studies have demonstrated thecross-protection among different arenaviruses, such as LCMV and PICV,Lassa and Mopeia virus, Junin and Tacaribe complex viruses, and thatapathogenic arenaviruses have been explored as live vaccines (reviewedin Olschlager et al., 2013, PLoS Pathog 9:e1003212). We propose that therP18tri vector incorporating protective T cell epitopes into PICVproteins can induce cross-protective immunity against other arenaviruspathogens. With a capacity of encoding up to two additional antigens,the rP18tri vector can be developed as a dual (or triple) vaccine vectoragainst both arenavirus pathogen and other desired pathogen(s).

In summary, a novel Pichinde virus (PICV)-based live viral vectorrP18tri was developed that packages 3 RNA segments and encodes 2 foreignprotein antigens. The viral vector was attenuated in vitro and in vivo.Using influenza HA as a model antigen, rP18tri-G/H can inducelong-lasting protective immunity in mice. The rP18tri vector can inducestrong humoral responses in mice and in guinea pigs and high levels ofvirus-specific effector T cells. The rP18tri-vector-induced antibody andT cell responses were significantly increased by a booster vaccinationand were at high levels even after four applications, a unique featureof this live viral vector that is ideal for a prime-and-boostvaccination strategy. The vector can be given via various routesincluding intramuscular (IM), intraperitoneal (IP), intranasal (IN), andoral. Priming and boosting with different HA proteins from differentinfluenza virus strains induced heterosubtypic immunity, thus there ispotential to develop universal flu vaccines. Chemically inactivatedrP18tri-P/H induced both humoral and CTL responses after a boost dose.The rP18tri vector induced protective immunity against virulent rP18virus, thus there is the potential for developing cross-reactivevaccines against other pathogenic arenaviruses such as Lassa fevervirus, and dual (triple) vaccines against combinations of otherpathogens.

The complete disclosure of all patents, patent applications, andpublications, and electronically available material (including, forinstance, nucleotide sequence submissions in, e.g., GenBank and RefSeq,and amino acid sequence submissions in, e.g., SwissProt, PIR, PRF, PDB,and translations from annotated coding regions in GenBank and RefSeq)cited herein are incorporated by reference in their entirety.Supplementary materials referenced in publications (such assupplementary tables, supplementary figures, supplementary materials andmethods, and/or supplementary experimental data) are likewiseincorporated by reference in their entirety. In the event that anyinconsistency exists between the disclosure of the present applicationand the disclosure(s) of any document incorporated herein by reference,the disclosure of the present application shall govern. The foregoingdetailed description and examples have been given for clarity ofunderstanding only. No unnecessary limitations are to be understoodtherefrom. The invention is not limited to the exact details shown anddescribed, for variations obvious to one skilled in the art will beincluded within the invention defined by the claims.

Unless otherwise indicated, all numbers expressing quantities ofcomponents, molecular weights, and so forth used in the specificationand claims are to be understood as being modified in all instances bythe term “about.” Accordingly, unless otherwise indicated to thecontrary, the numerical parameters set forth in the specification andclaims are approximations that may vary depending upon the desiredproperties sought to be obtained by the present invention. At the veryleast, and not as an attempt to limit the doctrine of equivalents to thescope of the claims, each numerical parameter should at least beconstrued in light of the number of reported significant digits and byapplying ordinary rounding techniques.

Notwithstanding that the numerical ranges and parameters setting forththe broad scope of the invention are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspossible. All numerical values, however, inherently contain a rangenecessarily resulting from the standard deviation found in theirrespective testing measurements.

All headings are for the convenience of the reader and should not beused to limit the meaning of the text that follows the heading, unlessso specified.

1-52. (canceled)
 53. A genetically engineered Pichinde virus comprising:three separate ambisense genomic segments, wherein the first genomicsegment comprises a coding region encoding a Z protein and a codingregion encoding a L RNA-dependent RNA polymerase protein, wherein thesecond genomic segment comprises a coding region encoding anucleoprotein (NP), wherein the third genomic segment comprises a codingregion encoding a glycoprotein wherein the second genomic segmentfurther comprises a coding region encoding a first protein, or the thirdgenomic segment further comprises a coding region encoding a secondprotein.
 54. The virus of claim 53 wherein the second genomic segmentfurther comprises the coding region encoding the first protein, and thethird genomic segment further comprises the coding region encoding thesecond protein.
 55. The virus of claim 53 wherein the first protein andthe second protein are selected from an antigen and a detectable marker.56. The virus of claim 53 wherein the first protein and the secondprotein encode an antigen.
 57. The virus of claim 55 wherein the antigenis a protein expressed by a viral pathogen, a prokaryotic pathogen, or aeukaryotic pathogen.
 58. The virus of claim 53 wherein the first proteinand the second protein are different.
 59. The virus of claim 53 whereinthe first protein and the second protein are the same.
 60. The virus ofclaim 53 wherein the nucleoprotein comprises at least one mutation thatreduces exoribonuclease activity of the nucleoprotein, wherein themutation is a mutation of at least one RNase catalytic residue.
 61. Thevirus of claim 53 wherein the glycoprotein comprises at least onemutation that alters the activity of the glycoprotein, wherein when theglycoprotein comprises SEQ ID NO:4 the mutation is selected from anasparagine at amino acid 20 and an asparagine at amino acid 404, or acombination thereof, and wherein the asparagine is substituted with anyother amino acid.
 62. An infectious virus particle comprising the threegenomic segments of claim
 53. 63. A composition comprising the isolatedinfectious virus particle of claim 62, wherein the infectious virusparticle is isolated.
 64. A collection of vectors comprising: a firstvector encoding a first genomic segment comprising a coding regionencoding a Z protein and a coding region encoding a L RdRp protein,wherein the coding region encoding the Z protein is antigenomic, asecond vector encoding a second genomic segment comprising a codingregion encoding a nucleoprotein (NP), wherein the coding region encodingthe NP is antigenomic, and a third vector encoding a third genomicsegment comprises a coding region encoding a glycoprotein, wherein thecoding region encoding the glycoprotein is antigenomic; and wherein thesecond genomic segment further comprises a coding region encoding afirst protein, or the third genomic segment further comprises a codingregion encoding a second protein.
 65. The collection of claim 64 whereinthe second genomic segment further comprises the coding region encodingthe first protein, and the third genomic segment further comprises thecoding region encoding the second protein.
 66. The collection of claim64 wherein the vectors are plasmids.
 67. The collection of claim 64wherein the plasmids further comprise a T7 promoter.
 68. The collectionof claim 64 wherein the first protein and the second protein areselected from an antigen and a detectable marker.
 69. The collection ofclaim 68 wherein the first protein and the second protein encode anantigen.
 70. The collection of claim 68 wherein the antigen is a proteinexpressed by a viral pathogen, a prokaryotic pathogen, or a eukaryoticpathogen.
 71. The collection of claim 64 wherein the first protein andthe second protein are different.
 72. The collection of claim 64 whereinthe first protein and the second protein are the same.
 73. A method formaking a genetically engineered Pichinde virus comprising: introducinginto a cell the collection of vectors of claim 64; and incubating thecells in a medium wherein the first, second, and third genomic segmentsare expressed and packaged.
 74. The method of claim 73 furthercomprising isolating an infectious virus particle from the medium. 75.The method of claim 73 wherein the cells express a T7 polymerase. 76.The isolated infectious virus particle of claim
 73. 77. A compositioncomprising the isolated infectious virus particle of claim
 76. 78. Areverse genetics system for making a genetically engineered Pichindevirus comprising three vectors, wherein a first vector encodes a firstgenomic segment comprising a coding region encoding a Z protein and acoding region encoding a L RdRp protein, wherein the coding regionencoding the Z protein is antigenomic, wherein the second vector encodesa second genomic segment comprising a coding region encoding anucleoprotein (NP), wherein the coding region encoding the NP isantigenomic, wherein the third vector encodes a third genomic segmentcomprises a coding region encoding a glycoprotein, wherein the codingregion encoding the glycoproteinis antigenomic wherein the secondgenomic segment further comprises a coding region encoding a firstprotein, or the third genomic segment further comprises a coding regionencoding a second protein.
 79. The reverse genetics system of claim 53wherein the second genomic segment further comprises the coding regionencoding the first protein, and the third genomic segment furthercomprises the coding region encoding the second protein.
 80. The reversegenetics system of claim 78 wherein each plasmid comprises a T7promoter.
 81. A method for using a reverse genetics system, comprising:introducing into a cell the three vectors of genomic segments of claim78; incubating the cell wherein the three genomic segments aretranscribed and the coding regions of each genomic segment areexpressed.
 82. The method of claim 81 further comprising isolatinginfectious virus particles produced by the cell, wherein each infectiousvirus particle comprises the three genomic segments.
 83. The method ofclaim 81 wherein the introducing comprises transfecting a cell with thethree genomic segments.
 84. The method of claim 81 wherein theintroducing comprises contacting the cell with an infectious virusparticle comprising the three genomic segments.
 85. The method of claim81 wherein the cell is ex vivo.
 86. The method of claim 81 wherein thecell is a vertebrate cell.
 87. The method of claim 86 wherein thevertebrate cell is a mammalian cell.
 88. The method of claim 87 whereinthe mammalian cell is a human cell.
 89. The method of claim 86 whereinthe vertebrate cell is an avian cell.
 90. The method of claim 89 whereinthe avian cell is a chicken embryonic fibroblast.
 91. A method forproducing an immune response in a subject, comprising administering to asubject the infectious virus particle of claim 62, wherein the secondgenomic segment further comprises a coding region encoding a firstantigen, or the third genomic segment further comprises a coding regionencoding a second antigen.
 92. The method of claim 91 wherein thesubject is a vertebrate.
 93. The method of claim 92 wherein thevertebrate is a mammal.
 94. The method of claim 93 wherein the mammalianis a human.
 95. The method of claim 91 wherein the vertebrate is avian.96. The method of claim 91 wherein the immune response comprises ahumoral immune response.
 97. The method of claim 91 wherein the immuneresponse comprises a cell-mediated immune response.
 98. The method ofclaim 91 wherein the first antigen or the second antigen is a proteinexpressed by a viral pathogen, a prokaryotic pathogen, or a eukaryoticpathogen, and wherein the subject is at risk of exposure to the viralpathogen, the prokaryotic pathogen, or the eukaryotic pathogen.
 99. Themethod of claim 91 wherein the administering comprises administering atleast two populations of infectious virus particles, wherein eachpopulation of infectious virus particle encodes a different antigen.